dapE gene of helicobacter pylori and dapE- mutant strains of helicobacter pylori

ABSTRACT

The invention provides the dapE gene of  Helicobacter pylori  and  H. pylon  dapE −  mutants and to methods of using the mutants to express foreign genes and immunize against foreign agents. The dapE gene can consist of the nucleotide sequence defined in SEQ ID NO:1. Nucleic acids of the gidA gene and ORF2 of  H. pylori  are provided. Examples of these nucleic acids can be found in SEQ ID NO:3 and SEQ ID NO:5, respectively. Having provided these nucleic acids, hybridizing nucleic acids in accord with the description of hybridizing nucleic acids of dapE are also provided.

[0001] This work was supported by NIHR01DK 50837. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention pertains to the dapE gene of Helicobacter pyloriand H. pylori dapE mutants and to methods of using the mutants toexpress foreign genes and immunize against foreign agents.

[0004] 2. Background Art

[0005]Helicobacter pylori are gram negative enteric bacteria thatcolonize the human gastric mucosa and cause gastritis and peptic ulcerdisease (6, 11, 15) and are implicated in malignant neoplasms of thestomach (5, 26, 30, 37). Thus, there exists a need for a method oftreating and preventing H. pylori infection.

[0006] The present invention meets these needs by providing the dapEgene of H. pylori and conditionally lethal mutants of H. pylori whichcan be used to express foreign proteins and to immunize against H.pylori infection.

BRIEF DESCRIPTION OF THE FIGURES

[0007]FIG. 1 shows physical maps of recombinant pBluescript plasmidscontaining gidA, dapE, and orf2. pAK1 contains the 3′ region of orf2plus approximately 4 kb downstream. pAK2 contains a 5 kb EcoRI fragmentthat includes gidA, dapE, and orf2. Boxes and arrows beneath theplasmids represent the location of the genes and the presumed directionof translation, and km represents a cassette encodingkanamycin-resistance. Arrowheads with numbers represent sites ofoligonucleotide primers used in PCR. Restriction endonuclease cleavagesites: Ba (BamHI), Bc (BclI), N (NdeI), E (EcoRI), and H (HindIII).

[0008]FIG. 2 shows the nucleotide and deduced amino acid sequences ofthe region on the H. pylori chromosome including gidA, and dapE, andorf2. SEQ ID NO:7 defines the nucleotide sequence shown in FIG. 2. Thesequence of gidA, dapE and orf2, and 579 bp upstream and 595 bpdownstream are shown. The 1866 bp gidA commences at nucleotide 580 andends at nucleotide 2445. The 1167 bp dapE commences at nucleotide 2456and ends at nucleotide 3622. The 753 bp orf2 commences at nucleotide3703 and ends at nucleotide 4455. The deduced amino acid sequence isshown beneath the nucleotides. A potential ribosome-binding site(Shine-Dalgarno sequence) and putative promoter elements (−35 and −10sequences) are indicated. An open reading frame, tentatively calledORF1, which is deduced to be translated in the opposite orientation fromgidA begins at nucleotide 483. An open reading frame, tentatively calledORF3, which is deduced to be translated in the opposite orientation fromorf2 ends at nucleotide 4655.

[0009]FIG. 3 shows alignment of the deduced amino acid sequences of thegidA (Panel A) and dapE (Panel B) products in E. coli and H. influenzaeand the respective H. pylori homologs. To optimize the alignments, gapswere introduced when necessary. The vertical lines between residuesindicate identity whereas two dots represents a conservativesubstitution.

DETAILED DESCRIPTION OF THE INVENTION

[0010] Nucleic Acids

[0011] An isolated dapE gene of Helicobacter pylori is provided.“Isolated” means a nucleic acid is separated from at least some of othercomponents of the naturally occurring organism, for example, the cellstructural components and/or other genes. The isolation of the nucleicacids can therefore be accomplished by techniques such as cell lysisfollowed by phenol plus chloroform extraction, followed by ethanolprecipitation of the nucleic acids (24). It is not contemplated that theisolated nucleic acids are necessarily totally free of non-nucleic acidcomponents, but that the isolated nucleic acids are isolated to a degreeof purification to be useful in a clinical, diagnostic, experimental, orother procedure such as gel electrophoresis, Southern or dot blothybridization, or PCR. A skilled artisan in the field will readilyappreciate that there are a multitude of procedures which may be used toisolate the nucleic acids prior to their use in other procedures. Theseinclude, but are not limited to, lysis of the cell followed by gelfiltration or anion exchange chromatography, binding DNA to silica inthe form of glass beads, filters or diatoms in the presence of highconcentration of chaotropic salts, or ethanol precipitation of thenucleic acids.

[0012] The nucleic acids of the present invention can include positiveand negative strand RNA as well as DNA and is meant to include genomicand subgenomic nucleic acids found in the naturally occurring organism.The nucleic acids contemplated by the present invention include doublestranded and single stranded DNA of the genome, complementary positivestranded cRNA and mRNA, and complementary cDNA produced therefrom andany nucleic acid which can selectively or specifically hybridize to theisolated nucleic acids provided herein.

[0013] The dapE gene can consist of the nucleotide sequence defined inSEQ ID NO:1. Other examples of the dapE gene of H. pylon can be found inany H. pylon isolate. Although there may be small differences (e.g.,point mutations) among the dapE genes of H. pylon strains, thesedifferences, if any, do not prevent the isolation and sequencing orother uses of this gene from other H. pylori strains. Thus, primers fromthe present dapE sequence can be used to amplify dapE from any sample inwhich it occurs, and oligonucleotide segments of the exemplified dapEgene can be used to probe a sample for the presence of the H. pyloridapE or, more generally, H. pylori. It may be preferable to use slightlylonger primers than the standard primers of 17 nucleotides, for example,primers of approximately 25 nucleotides.

[0014] The dapE gene can be distinguished from other nucleic acids,because of its conserved genomic location. Particularly, dapE is flankedupstream by gidA and downstream by orf2. This conserved location alsomakes obtaining dapE from other H. pylon strains routine andpredictable. For example, primers that hybridize with the highlyconserved gidA and orf2 can be used to amplify dapE from any sample inwhich it occurs. Similarly, a primer that hybridizes with one or theother of the highly conserved gidA and orf2 can be paired with a primerfrom the exemplified dapE to amplify dapE (or a segment of it) from anysample in which it occurs. Additionally, since the position of this genein the H. pylori genome is known, it can be mutated in any strain,according to the methods taught herein.

[0015] DapE-encoding nucleic acids can be isolated from H. pylori) usingany of the routine techniques. For example, a genomic DNA or cDNAlibrary can be constructed and screened for the presence of the nucleicacid of interest using one of the present dapE nucleic acids as a probe.Methods of constructing and screening such libraries are well known inthe art and kits for performing the construction and screening steps arecommercially available (for example, Stratagene Cloning Systems, LaJolla, Calif.). Furthermore, genomic DNA can be isolated from an H.pylori strain and screened using one of the present dapE nucleic acidsas a probe. Once isolated, the dapE nucleic acid can be directly clonedinto an appropriate vector, or if necessary, be modified to facilitatethe subsequent cloning steps. Such modification steps are routine, anexample of which is the addition of oligonucleotide linkers whichcontain restriction sites to the termini of the nucleic acid. Generalmethods are set forth in Sambrook et al. (24).

[0016] A H. pylori-specific nucleic acid fragment of the dapE gene isprovided. For example, the fragment can consist of the nucleotidesequence of the 1.1 kb dapE-specific fragment, further described in theExamples. Other examples can be obtained routinely using the methodstaught herein and in the art.

[0017] A nucleic acid that encodes a naturally occurring DapE protein ofHelicobacter pylori and hybridizes with the nucleic acid of SEQ ID NO:1under the stringency conditions of about 16 hrs at about 65° C., about5×SSC, about 0.1% SDS, about 2× Denhardt's solution, about 150 μg/mlsalmon sperm DNA with washing at about 65° C., 30 min, 2×, in about0.1×SSPE/0.1% SDS is provided. Alternative hybridization conditionsinclude. 68° C. for about 16 hours in buffer containing about 6×SSC,0.5% sodium dodecyl sulfate, about 5× Denhardt's solution and about 100μg salmon sperm DNA, with washing at about 60° C. in about 0.5×SSC(Tummuru, M. K. R., T. Cover, and M. J. Blaser (24).

[0018] A nucleic acid probe that hybridizes with the nucleic acid of SEQID NO:1 under either of the above described stringency conditions can beused to identify dapE in other strains of H. pylori.

[0019] A nucleic acid primer that hybridizes with the nucleic acid ofSEQ ID NO:1 under the polymerase chain reaction conditions of 35 cyclesof 94° C. for 1 min, 50° C. for 2 min, and 72° C. for 2 min, with aterminal extension at 72° C. for 10 min. These conditions can be usedwith the relevant primers to identify dapE, including interstrainvariants. Examples of these primers are described in the Examples.

[0020] The selectively hybridizing nucleic acids of the invention canhave at least 70%, 80%, 85%, 90%, 95%, 97%, 98% and 99% complementaritywith the segment and strand of the sequence to which it hybridizes. Thenucleic acids can be at least 15, 18, 20, 25, 50, 100, 150, 200, 300,500, 750, 1000, 2000, 3000 or 4000 nucleotides in length. Thus, thenucleic acid can be an alternative coding sequence for the H. pyloridapE, or can be used as a probe or primer for detecting the presence H.pylori. If used as primers, the invention provides compositionsincluding at least two nucleic acids which selectively hybridize withdifferent regions so as to amplify a desired region. Depending on thelength of the probe or primer, it can range between 70% complementarybases and full complementarity with the segment to which it hybridizes.For example, for the purpose of diagnosing the presence of H. pylori,the degree of complementarity between the hybridizing nucleic acid(probe or primer) and the sequence to which it hybridizes (H. pylori DNAfrom a sample) should be at least enough to exclude hybridization with anucleic acid from related bacterium. Thus, a nucleic acid thatselectively hybridizes with a H. pylori DapE coding sequence will notselectively hybridize under stringent conditions with a nucleic acid fora DapE of another species, and vice versa. The degree of complementarityrequired to distinguish selectively hybridizing from nonselectivelyhybridizing nucleic acids under stringent conditions can be clearlydetermined for each nucleic acid. It should also be clear that aselectively hybridizing nucleic acid will not hybridize with nucleicacids encoding unrelated proteins.

[0021] Nucleic acids of the gidA gene and ORF2 of H. pylori areprovided. Examples of these nucleic acids can be found in SEQ ID NO:3and SEQ ID NO:5, respectively. Having provided these nucleic acids,hybridizing nucleic acids in accord with the description of hybridizingnucleic acids of dapE are also provided.

[0022] The nucleic acids of the invention (dapE, gidA and ORF2, andtheir fragments) can also be in a composition such as a geneticconstruct, which includes other nucleic acids such as origins ofreplication, promoters, other protein coding sequences, etc. Thecomposition can also, or alternatively, contain compounds, such asnon-nucleic acid marker molecules attached to the nucleic acids.

[0023] Vectors

[0024] The DapE-encoding nucleic acid and selectively hybridizingnucleic acids of the invention can be in a vector suitable forexpressing the nucleic acid. The nucleic acid in a vector can be in ahost suitable for expressing the nucleic acid.

[0025] A plasmid comprising a nucleic acid encoding a functional DapEprotein of H. pylori is provided. The plasmid can further comprise anucleic acid encoding a non-H. pylori (foreign) protein. The foreignprotein encoded can be immunogenic, antigenic, or enzymatic proteins ofbacteria, virus, fungus or parasite. For example the protein can be fromSalmonella (Salmonella enteritidis), Shigella species, Yersinia,enterotoxigenic and enterohemorrhagic E. coli, Mycobacteriumtuberculosis, Streptococcus pyogenes, Bordatella pertussis, Bacillusanthracis, P. falciparum, human immunodeficiency virus, respiratorysyncytial virus, influenza virus, histoplasma capsulatum or otherinfectious organisms.

[0026] Other foreign proteins include sperm antigens for use to immunizeagainst sperm as a form of birth control. Also contemplated areimmunomodulating proteins to treat against inflammatory diseases andcytotoxic proteins to treat against malignancies. Likewise, the foreignprotein need not be an antigen, but can instead be protein of the host.In this embodiment the H. pylon mutant strain is used to express a hostprotein in vivo to provide or augment an activity of the host protein.Thus, the H. pylori dapE mutant can include an insulin gene for use in amethod of delivering insulin to diabetics or it can include a gene forTNF for modulating or augmenting the host response to stress. In eachcase the amount of DAP provided to the host can be used to control theamount of the protein that is expressed. As noted elsewhere in theapplication, the H. pylori dapE strain can also have other introducedattenuating mutations or it can be a naturally occurring vacuolatingtoxin⁻ strain.

[0027] The plasmids of the present invention can be any of the well knowplasmids used in bacteria. For example, the plasmid can include a 1.5 kbHindIII fragment cloned into the polylinker of a pUC vector. One suchvector has a kanamycin resistance cassette inserted into this hybridclone and is called pHP1.

[0028] Another shuttle vector that can be used as an example of the typeof construct that would be useful, was that described by Schmitt et al.(Mol. Gen. Genetics. 1995. 248:563-472). In the case described, acryptic H. pylori plasmid, pHe1 was ligated with an E. coli replicon toprepare an E. coli-H. pylori shuttle vector. Schmitt et al. cloned theH. pylori recA gene into the shuttle vector to create pDH38 which wasintroduced into an H. pylori strain by natural transformation. Thisvector was shown to complement the recA deficiency on a parental strain.

[0029] Vectors other than plasmids that can be used in H. pylori includetransducing bacteriophage.

[0030] Mutant H. pylori

[0031] A purified mutant strain of H. pylori that does not express afunctional DapE protein is provided. The mutant can either not expressDapE or express a non-functioning DapE. Such a mutant is also referredto herein as a dapE⁻ mutant. Mutations to the dapE gene on the H. pylorichromosome that result in a dapE⁻ mutant can be made by insertion,deletion or internal modification.

[0032] In one example, the mutant H. pylori strain is obtained by makingan insertion substitution mutation in the coding sequence for the DapEas described in the Examples. Since the present invention provides thenucleic acid encoding DapE, other methods of mutating the codingsequence of DapE can be used to obtain other mutant strains ascontemplated herein.

[0033] One example of the dapE⁻ mutant strain is deposited with theAmerican Type Culture Collection, 12301 Parklawn Drive Rockville, Md.20852 on Dec. 6, 1996 under accession no. ATCC 55897. This example wasmade according to one method of making such mutants taught in theExamples.

[0034] Additional mutants can be prepared, for example, by inserting anucleic acid in the dapE gene or deleting a portion of the dapE gene soas to render the gene non-functional or protein produced in such lowamounts that the organism dies in the absence of DapE supplementation.Furthermore, by providing the nucleotide sequence for the nucleic acidencoding the DapE, the present invention permits the making of specificpoint mutations having the desired effect. The deletion, insertion orsubstitution mutations can be made in either the regulatory or codingregion to prevent transcription or translation or to render thetranscribed and translated product nonfunctional.

[0035] One such approach to the construction of a deletion or insertionmutant is via the Donnenberg method (Donnenberg and Kaper Infect. Immun.4310-4317, 1991). A deletion in dapE is created by deleting arestriction fragment and religating the clone. This mutant is clonedinto suicide vector pILL570. The sacB gene of Bacillus subtilis is alsocloned into the suicide vector to provide a conditionally lethalphenotype. This construct is transformed into H. pylori byelectroporation, and transformants selected by spectinomycin resistance.The merodiploid strain which contains the suicide vector and the mutatedversion of the dapE gene are exposed to sucrose to directly select fororganisms that have undergone a second recombination, resulting in theloss of the vector. These and other well known methods of makingmutations can be applied to the nucleic acids provided herein to obtainother desired mutations. Included are insertional mutagenesis asdescribed in reference 8, as well as linker-scanning mutagenesis (46)and site-directed mutagenesis (47).

[0036] Non-isogenic mutants are also within the scope of the invention.For example, a live attenuated H. pylori that is also a dapE⁻ mutantaccording to the present invention, is provided. A dapE⁻recA⁻ mutantstrain is constructed, for example, by insertion mutation of both thedapE and recA genes, according to the methods taught herein for dapE andin U.S. Pat. No. 5,434,253, issued on Jul. 18, 1995 for recA. AdapE⁻recA-cagA-mutant strain is constructed, for example, by insertionmutation of all three genes, according to the methods taught herein, inU.S. Pat. No. 5,434,253 and in U.S. application Ser. No. 08/053,614,which describes the generation of a cagA (referred to therein as tagA)mutant. A dapE⁻recA⁻vacA⁻ mutant strain is constructed, for example, byinsertion mutation of all three genes, according to the methods taughtherein. A dapE⁻recA⁻cagA⁻vacA⁻ mutant strain is constructed, forexample, by insertion mutation of all four genes, according to themethods taught herein and the above cited patents and patentapplications. Furthermore, a mutation in dapE combined with any one ormore of the above or other mutations can be made. Any of the well knownmethods of mutating a gene can be used in the present invention togenerate H. pylori mutant strains. The strains can be tested as providedfor immunogenicity, conditional lethality, vacuolating activity, etc.

[0037] The dapE⁻ mutant strain can also have in its chromosome a nucleicacid encoding a foreign protein. Briefly, this can be accomplished byinserting SacB in the chromosome as above, then using a suicide vectorthat has the foreign gene replacing dapE and flanked by gidA and orf2.The suicide vector is then transformed into H. pylon using naturaltransformation conditions such as described in U.S. Pat. No. 5,434,253,and in U.S. application Ser. Nos. 08/053,614, 08/215,928 and 08/200,232.After transformation the H. pylori is then grown on sucrose-containingplates. This selects for replacement of the sacB insert by the foreigngene.

[0038] The foreign protein encoded can be as described above.

[0039] The dapE⁻ mutant strain can also contain a plasmid comprising anucleic acid encoding a foreign protein. The foreign protein encoded canbe as described above.

[0040] The dapE⁻ mutant H. pylori can be transformed with, and thus,contain a plasmid comprising a nucleic acid encoding a functional DapEprotein. This can be accomplished using natural transformation such asis described in U.S. Pat. No. 5,434,253, and in U.S. application Ser.Nos. 08/053,614, 08/215,928 and 08/200,232. Under the appropriateconditions the dapE⁻ mutant H. pylon can express a functioning dapEprotein due to trans complementation by the dapE gene on the plasmid.These are the typical growth conditions for H. pylori, such as aredescribed in the example.

[0041] The mutant H. pylori containing a plasmid comprising a nucleicacid encoding a functional DapE protein can include in its chromosome aforeign nucleic acid encoding a foreign protein. A method of inserting aforeign gene in the H. pylon chromosome is described above. The mutantH. pylori containing a plasmid comprising a nucleic acid encoding afunctional DapE protein wherein the plasmid further comprises a nucleicacid encoding a foreign protein is provided.

[0042] Having provided the present conditionally lethal dapE⁻ mutant andmethod of generating such a conditionally lethal mutation in the genomeof H. pylori, the present invention leads predictably to otherconditionally lethal mutations in H. pylori. Such mutants include thosewith mutations in genes essential for cell wall synthesis or particularbiochemical pathways for which the product can be used to complement themutation.

[0043] The dapE⁻ mutant strain containing a foreign protein in eitherits chromosome or in a plasmid can be used as an expression system forexpressing the foreign protein.

[0044] Foreign Gene Expression Method

[0045] The present invention provides methods to deliver foreignantigens via the present dapE⁻ mutant H. pylori strain engineered toalso contain nucleic acids either in the chromosome or on a plasmid(e.g., an H. pylori shuttle plasmid) which express the foreign antigenof interest.

[0046] A method is provided for maintaining the expression of a foreignantigen in Helicobacter pylori, comprising: a) transforming a mutantHelicobacter pylori that does not produce of functioning DapE proteinwith a plasmid comprising a nucleic acid encoding a functional DapEprotein and comprising a nucleic acid encoding the foreign protein; andb) maintaining the mutant Helicobacter pylon from step a underconditions that permit expression of the foreign protein. Furthermore,by maintaining the H. pylori from step a in medium without added L-DAP,only the H. pylori that contain the plasmid will survive.

[0047] Screening for H. pylori Mutants

[0048] The present dapE mutant can be used in a method of screening forand selecting H. pylori mutants. Because of the attributes of the dapE⁻mutant, mutants can be selected without the use of antibioticresistance. This makes the mutants safer for use in humans, becausethere is no risk of introducing an organism that is resistant toantibiotics, and thus, not removable by antibiotic treatment. Briefly,dapE is deleted from the chromosome or insertionally mutated and thestain is grown in L-DAP-containing medium. A shuttle vector (plasmid) isconstructed that encodes DapE to complement the dapE mutation in thechromosome and encodes a foreign protein. The plasmid is transformedinto the dapE⁻ H. pylori, which are grown on medium without L-DAP. Thisselects for maintenance of the plasmid.

[0049] It should be noted that in the methods and compositionsdescribed, the foreign antigen gene can either be in the chromosomereplacing dapE or elsewhere in the chromosome. The foreign gene can beon the plasmid that encodes DapE, so that dapE⁻ strains can be used ashosts for any number (multiple copies) or type (cocktail) of gene thatmay be on any number of plasmids. These strains are expected to survivewell since functional DapE is provided. They can be attenuatedelsewhere, for example in recA or in vacA so that they are less toxic.

[0050] Immunization Methods

[0051] An immunogenic amount of the dapE⁻ mutant H. pylori in apharmaceutically acceptable carrier can be used as a vaccine.

[0052] A method is provided for immunizing a subject against infectionwith Helicobacter pylori, comprising: a) administering to the subjectthe dapE⁻ mutant strain of H. pylori; b) supplementing the subject'sdiet with diaminopimelic acid (DAP) in the form of L-DAP or meso DAP (amixture of L-DAP and D-DAP) to maintain the mutant strain in the subjectat least long enough for the subject to mount an immune response to thestrain; and c) ceasing the supplementation of the subject's diet withdiaminopimelic acid to kill the mutant strain in the immunized subject.The immunization methods described herein comprise administering to thesubject an immunogenic amount of mutant H. pylori in a pharmaceuticallyacceptable carrier for the mutant. The length of time to which thesubject is exposed to the mutant strain, i.e., the length of time L-DAPsupplementation is provided, will typically be from a few days (2 or 3)up to a few weeks (2 or 3). The exact time course may vary fromindividual to individual and can be verified by tests for the presenceof an immune response such as indicated by the presence of antibodiesagainst the H. pylon in a tissue sample (e.g., gastric juice, blood,plasma, urine and saliva) from the subject.

[0053] A method of immunizing a subject against bacterial infection isprovided, comprising: a) administering to the subject the dapE⁻ mutantstrain having in its chromosome a nucleic acid encoding a foreignprotein (e.g., an immunogen of the bacterium); b) supplementing thesubject's diet with L-DAP to maintain the mutant strain in the subjectat least long enough for the subject to mount an immune response to thestrain; and c) ceasing the supplementation of the subject's diet withdiaminopimelic acid to kill the mutant strain in the immunized subject.The immunization methods described herein comprise administering to thesubject an immunogenic amount of mutant H. pylori in a pharmaceuticallyacceptable carrier for the mutant. The length of time to which thesubject is exposed to the mutant strain is as described above. The exacttime course will vary from individual to individual and can be verifiedby tests for the presence of an immune response such as is measured byassays for antibodies against the foreign protein in a tissue sample(e.g., gastric juice, blood, plasma, urine and saliva) from the subject.

[0054] A method of immunizing a subject against a bacterial infection,comprising: a) administering to the subject the dapE⁻ mutant straincontaining a plasmid comprising a nucleic acid encoding a foreignprotein; b) supplementing the subject's diet with diaminopimelic acid tomaintain the mutant strain in the subject at least long enough for thesubject to mount an immune response to the strain; and c) ceasing thesupplementation of the subject's diet with diaminopimelic acid to killthe mutant strain in the immunized subject.

[0055] In the above methods wherein the foreign antigen encoding gene ison the dapE-containing plasmid, the host strain can be attenuated inother genes (e.g., recA, vacA, etc.)

[0056] The present immunization methods are useful in immunizing againstinfection with bacteria, fungi, protozoa and viruses. Thus, the foreignprotein encoded can be an antigen (immunogen) of the bacterium, virus,fungus or protozoan against which immunization is being made. Theforeign protein encoded can be immunogenic, antigenic, or enzymaticproteins of Salmonella (Salmonella enteritidis), Shigella, Yersinia,enterotoxigenic and enterohemorrhagic E. coli, M. tuberculosis,Streptococcus pyogenes, P. falciparum, human immunodeficiency virus,respiratory syncytial virus, influenza virus or other infectiousorganisms. Other foreign proteins include sperm antigens for use toimmunize against sperm as a form of birth control or immunomodulatingproteins or cytotoxic proteins as described above. The immunizationmethods for these microorganisms also comprise administering to thesubject an immunogenic amount of mutant H. pylori containing the genefor a foreign immunogen in a pharmaceutically acceptable carrier for themutant. The length of time to which the subject is exposed to the mutantstrain, i.e., the length of time L-DAP supplementation is provided, willtypically be from a few days (2 or 3) up to a few weeks (2 or 3). Theexact time course may vary from individual to individual and can beverified by tests for the presence an of immune response (e.g., by thepresence of antibodies) against the foreign protein in a tissue sample(gastric juice, blood, plasma, urine and saliva) from the subject.Clearly, if the subject produces antibodies against the microorganism,it is understood that the antibodies are against the recombinant proteinproduced by the altered H. pylori strain.

[0057] Determining Immunogenicity and Immunogenic Amounts

[0058] The isolated mutant strains of the invention can be tested todetermine their immunogenicity. Briefly, various concentrations of aputative immunogen are prepared and administered to an animal and theimmunological response (e.g., the production of antibodies or cellmediated immunity) of an animal to each concentration is determined. Theamounts of antigen administered depend on the subject, e.g. a human,mouse or gerbil, the condition of the subject, the size of the subject,etc. Thereafter, an animal so inoculated with the strain can be exposedto the bacterium to test the potential vaccine effect of the specificimmunogenic protein or fragment.

[0059] For example, well-established models include gnotobiotic pigletsand mice. The dapE⁻ mutant strain is first fed to the piglets or miceand maintained by DapE supplementation. After a suitable interval, thesupplementation is stopped and the clearance of the vaccine strain isevaluated. Next, this piglet or mouse is challenged with a wild-type H.pylori strain or other microorganism and the presence or absence ofinfection is ascertained (48, 49). This same system can also be used todetermine the amounts of mutant required to have a protective effect.

[0060] Once immunogenicity is established as described above,immunogenic amounts of the antigen can be determined using standardprocedures. Briefly, various concentrations of the dapE⁻ mutant areprepared, administered to an animal and the immunological response(e.g., the production of antibodies) of an animal to each concentrationis determined.

[0061] Pharmaceutically Acceptable Carrier

[0062] The pharmaceutically acceptable carrier in the vaccine of theinstant invention can comprise saline or other suitable carriers (Arnon,R. (Ed.) Synthetic Vaccines I:83-92, CRC Press, Inc., Boca Raton, Fla.,1987). An adjuvant can also be a part of the carrier of the vaccine, inwhich case it can be selected by standard criteria based on the antigenused, the mode of administration and the subject (Arnon, R. (Ed.),1987). Methods of administration can be by oral or sublingual means, orby injection, depending on the particular vaccine used and the subjectto whom it is administered.

[0063] It can be appreciated from the above that the vaccine andimmunization method can be used as a prophylactic or a therapeuticmodality, for example, by inducing a therapeutic immune response. Thus,the invention provides methods of preventing or treating H. pyloriinfection and the associated diseases by administering the vaccine to asubject. Because the dapE⁻ mutant can contain and express many differentforeign immunogens, the invention also provides methods of treating orpreventing infections with other organisms.

[0064] DapE, GidA and ORF2 Proteins

[0065] A purified DapE protein of Helicobacter pylori is provided. TheDapE protein can consist of the amino acid sequence defined in SEQ IDNO:2. A H. pylori-specific fragment of the DapE protein can be routinelyobtained in accord with teaching herein and in the art, and iscontemplated.

[0066] Similarly, the GidA protein and the protein encoded by ORF2 areprovided in SEQ ID NOS:4 and 6.

EXAMPLES

[0067] Characterization of Helicobacter pylori dapE and Construction ofa Conditionally Lethal dapE⁻ Mutant

[0068] Bacterial Strains, Plasmids and Growth Conditions.

[0069]H. pylon strain 60190 was used for the molecular cloning studies,and 21 well characterized clinical H. pylori strains from the VanderbiltUniversity Helicobacter/Campylobacter culture collection were used todetermine the conservation of the cloned genes. Stock cultures weremaintained at −70° C. in Brucella Broth (BBL Microbiology Systems,Cockysville, Md.) supplemented with 15% glycerol. E. coli DH5α,XL-1blue, and Dam⁻ strains were used for transformation, and pBluescript(Stratagene, La Jolla, Calif., USA) was used as a cloning vector. E.coli strains were routinely cultured in Luria-Bertani (LB) medium withshaking at 37° C., and the clinical H. pylori isolates were cultured onTrypticase soy agar plates containing 5% sheep blood in a microaerobicatmosphere, as described (13). For transformation of H. pylori (14),strains were grown at 37° C. in a microaerobic atmosphere on Brucellaagar plates containing 5% Fetal Calf Serum (FCS) and 30 μg/ml kanamycinand supplements of 0 to 2 mM DAP (a racemic mixture of all three DAPisomers, Sigma Chemical Co, St. Louis, Mo.) or 1 mM lysine (Sigma).

[0070] Genetic Techniques and Nucleotide Sequence Analysis.

[0071] Chromosomal DNA was prepared as described previously (40). Allother standard molecular genetic techniques including Southern andcolony hybridizations were performed, as described (24,39). Formolecular cloning, positive plaques were purified from a bank ofapproximately 5 kb random chromosomal fragments of H. pylori 60190 usingZapII and recombinant DNA was prepared as described (40). Restrictionenzyme cleavage maps were generated, and a 5 kb fragment was subclonedinto pBluescript to create pAK2 (FIG. 1). Another Skb fragment carryinga portion of the H. pylori genome overlapping only the orf2 region ofpAK2 was subcloned into pBluescript to create pAK1 (FIG. 1). Thenucleotide sequence was determined unambiguously on both strands usingdouble-stranded DNA templates using an automated DNA sequencer (PerkinElmer, Model ABI377, Foster City, Calif.) with the ThermoSequenase dyeprimer reaction kit (Amersham, Arlington Heights, Ill.). Oligonucleotideprimers were synthesized at the Vanderbilt Cancer Center DNA corefacility with an ABI 392 DNA synthesizer sequencer (Perkin Elmer).Nucleotide sequences were compiled and analyzed using programs in theGCG Package (16). Amplifications were conducted in a Perkin-ElmerThermal Cycler. PCR conditions used in this study were 35 cycles of 94°C. for 1 min, 50° C. for 2 min, and 72° C. for 2 min, with a terminalextension at 72° C. for 10 min, and the primers used in this study arelisted in Table 1. TABLE 1 PCR Primers used in this study DesignationGene Position^(a) Strand Length Sequence Reference 1 gidA 569-586 + 18CAGGAAAAAGAGTGGTAA (SEQ ID No: 8) This work 2 gidA 2428-2445 − 18TTAAGAGTTTTTTCGCAA (SEQ ID No: 9) This work 3 dapE 2445-2462 + 18AAGGATATTTAATGAACG (SEQ ID No: 10) This work 4 dapE 3613-3633 − 21GTTTATTTATTTTATGCCTCA (SEQ ID No: 11) This work 5 orf2 3801-3819 + 19TAATTTAGGCATAGAGAGC (SEQ ID No: 12) This work 6 orf2 4024-4044 + 20TATAACGGACAAGGCGTATCT (SEQ ID No. 13) This work 7 orf2 4429-4450 − 24GTTCTATTTTCAATTCCTTGAGAG (SEQ ID No. 14) This work 8 orf3 5086-5103 − 18GCGTGAATGAATACGATA (SEQ ID No. 15) This work 9 km 689-712 − 24CTCCCACCAGCTTATATACCTTAG (SEQ ID No. 16) 22 10 km 1336-1356 + 21CTGGGGATCAAGCCTGATTGG (SEQ ID No. 17) 22 11 km 572-591 − 20GACCGTTCCGTGGCAAAGCA (SEQ ID No. 18) 38 12 km 1601-1622 + 22CTTGTGCAATGTAACATCAGAG (SEQ ID No. 19) 38 13 gidA 2300-2317 + 18GCATTCCAGGCTTAAGCT (SEQ ID No. 20) This work 14 dapE 2631-2650 − 20TGCATGTTCTTTTTCTGCAT (SEQ ID No. 21) This work 15 dapE 3506-3523 + 18GAGTTTGGCGTTATTAAT (SEQ ID No. 22) This work 16 orf2 3850-3866 − 17GCTTTTTCAAAATGCGT (SEQ ID No. 23) This work 17 vacA 4116-4134 − 16AAGCTTGATCACTCC (SEQ ID No. 24) 14

[0072] Construction of Recombinant Plasmids with Insertion ofKanamycin-Resistance Cassettes into Targeted Genes.

[0073] A C. coli km gene (22) was ligated into the unique BclI site ofpAK2 within the gidA ORF to create pAK2:gidA:km (pME36) (FIG. 1). An E.coli km cassette from pUC4K (38) was inserted into the unique NdeI sitewithin the dapE ORF to create pAK2:dapE:km (pMAK36) (FIG. 1). orf2contained no unique sites for km insertion, but 3 HindIII sites werepresent within 107 bp. Therefore, to create orf2:km, the orf2 ORF frompAK1 was PCR-amplified and subcloned the amplified fragment into pT7Blue(Novagen, Madison, Wis.) to create pAK7. The 430 bp insert was subclonedinto pCR-Script Cam SK (+), (Stratagene, La Jolla, Calif.), apBluescript derivative encoding chloramphenicol resistance, to createpAK8. After HindIII digestion of pAK8, the km cassette from pUC4K wasinserted into orf2 to create pAK8:orf2:km (pAKQ) (FIG. 1).

[0074] Construction of H. pylori dapE and orf2 Mutants.

[0075] The constructs, pAK2:gidA:km (pME36), pAK2:dapE:km (pMAK36), orpAK8:orf2:km (pAKQ), all of which are unable to replicate in H. pylori,were introduced into H. pylori 60190 by natural transformation; pCTB8:kmcontaining vacA:km was used as a positive control (14). Thetransformants were selected on Brucella broth agar plates containing 5%FCS and 30 μg/ml kanamycin. In certain experiments, plates weresupplemented with 1 mM DAP to determine the conditions necessary fordapE⁻ mutant viability. To determine the minimum concentration neededfor growth of the dapE⁻ mutant, strains were grown on media supplementedwith 0 to 2.0 mM DAP or 1.0 mM lysine. To provide genetic evidence inthe transformed strain of dapE disruption by the km insertion, DNAisolated from both the H. pylori mutant strain 60190 pAK2:dapE:km andwild-type strain 60190 was digested with BamHI and hybridized to dapEand km probes. The authenticity of the mutant strain also was verifiedby PCR, using primers based on dapE and km (FIG. 1 and Table 1). Theauthenticity of the orf2⁻ mutants also was verified by Southernhybridization and PCR using parallel methods.

[0076] Evidence of Homologous Recombination Between pAK2:gidA:km and H.pylori Strain 60190 Chromosomal DNA.

[0077] No viable gidA mutants were obtained, even with selection onmedia supplemented with DAP or lysine. To provide genetic evidence thatdouble cross-over events had occurred during the pre-selective growthphase, allowing for the insertion of km in the H. pylori chromosomewithin gidA, PCR was performed with a forward primer specific for km(primer 10 in FIG. 1 and Table 1) and a reverse primer (primer 8 in FIG.1 and Table 1) specific for a region of the H. pylori chromosome presentin pAK1 that is beyond the fragment cloned in pAK2. DNA isolated fromwild type strain 60190 was examined after overnight incubation withpAK2:gidA:km. As negative controls, DNA from wild type strain 60190 anda mixture of DNA from wild type strain 60190 and pAK2:gidA:km were used.As a positive control, the forward km primer (primer 10 in FIG. 1 andTable 1) and a confirmed vacA reverse primer (primer 17 in Table 1) (14)were tested on DNA isolated from wild type strain 60190 after overnightincubation with pCTB8:vacA:km.

[0078] RNA Isolation, Reverse Transcriptase-Polymerase Chain Reaction(RT-PCR), and Slot-Blot Analysis.

[0079] To determine whether gidA, dapE, and orf2 are co-transcribed,wild type and mutant H. pylori strains were cultured for 24 h onBrucella agar plates containing 5% FCS supplemented with 1 mM DAP, cellswere harvested, and RNA was recovered for RT-PCR by two rounds ofhot-phenol extraction, as described previously (40). cDNA wassynthesized from lag of DNAse-treated total RNA by priming with 1 μg ofrandom hexamer (Pharmacia, Inc., Piscataway, N.J.), 1 mM of each dNTP,20 units of RNAse inhibitor and AMV reverse transcriptase (Promega,Madison, Wis.) in a final volume of 20 ul at 42° C. for 15 min. PCRreactions were performed as described above.

[0080] Agarose gel electrophoresis was performed on specific RT-PCRamplified products from H. pylori wild-type and mutant strains usingprimers within gidA, dapE, or orf2 as follows: Lanes 1, 4, 7-wild typestrain 60190; lanes 2, 5, 8-dapE⁻ mutant strain (60190E⁻); lanes 3, 6,9-orf2⁻ mutant strain (60190-2⁻). PCR was performed using primers 13 and14 and DNA (lanes 1-3), cDNA (lanes 4-6) or RNA (lanes 7-9) astemplates. PCR was performed using primers 15 and 16 and DNA (lanes1-3), cDNA (lanes 4-6) or RNA (lanes 7-9) as templates.

[0081] To provide further evidence that orf2 is co-transcribed withdapE, slot-blot RNA analysis of mRNA transcripts of gidA, dapE, and orf2was used. DNAse-treated RNA samples (12 μg) from wild type (WT) H.pylori strain 60190, or its dapE⁻ or orf2⁻ mutants were transferred tonylon membranes, and hybridized with equal amounts (50,000 cpm) ofradiolabelled cagA, gidA, dapE, or orf2 probes. Hybridization usedprobes specific for gidA (1.9 kb PCR-amplified gidA-specific fragment),dapE (10.1 kb PCR-amplified dapE-specific fragment), orf2 (0.7 kbPCR-amplified orf2-specific fragment), or cagA as positive control (0.5kb PCR-amplified cagA-specific fragment). The amount of radiolabel(50,000 cpm) was standardized for each probe, and experiments wereperformed as previously described (32).

[0082] Isolation of H. pylori dapE.

[0083] A 5 kb EcoRI genomic fragment from H. pylori strain 60190 wascloned into pBluescript to create pAK2 (FIG. 1), and the nucleotidesequence of this fragment was determined (FIG. 2). Analysis oftranslation of the 5050 bp nucleotide sequence in all possible readingframes revealed five complete or partial open reading frames (ORFs). Thethree complete ORFs, consisting of 1866, 1167, and 753 nucleotides, wereoriented in the same direction and opposite to the partial ORFs presentat the ends of the fragment (FIGS. 1 and 2). The first complete ORFbegins with GTG as the initiation codon, and encodes a 621 amino acidpolypeptide, yielding a predicted product with a 69,665 Da molecularmass. The second ORF begins with an ATG codon, 10 bp after thetermination of the first ORF and encodes a 388 amino acid polypeptide,yielding a predicted 42,822 Da product. The third ORF begins with ATG 80bp after the termination of the second ORF, and encodes a 250 amino acidpolypeptide with a predicted molecular mass of 27,585 Da. Potentialribosome binding sites begin 6 or 7 bp upstream of each ORF. Upstream ofthe translational start of the first ORF is the sequence TATTTT, whichresembles the consensus σ70-10 sequence (33), and is 19 bp downstream ofthe sequence TTGGCA that shares 5 of 6 bases with the corresponding −35consensus sequence (33). Nucleotides 4456 to 4654 following the thirdORF exhibit the sequence of a putative three-hairpin stem-loop structure(ΔG=−40.2) that could permit a strong mRNA transcriptional terminator.The single putative promoter and transcription terminator and the closelocation and orientation of the ORFs suggest that they may represent anoperon.

[0084] Analysis of the Deduced Products of the ORFs.

[0085] The translated amino acid sequence for genes in pAK2 was comparedwith databases using the FASTA, FASTDB, and BLAST network services ofthe National Center for Biotechnology Information. The deduced productfrom the first complete ORF showed significant homology throughout thetranslated amino acid sequence (48.3% identity and 66.5% similarity)with the glucose inhibited division protein, encoded by gidA in E. coli(9,36,43), H. influenzae (47.1% identity and 67.5% similarity) (18)(FIG. 3A), Pseudomonas putida (28) (47.9% identity and 68.9%similarity), and Bacillus subtilis (27,28) (46.1% identity and 64.4%similarity). The putative product of the second ORF showed significanthomology (37.9% identity and 61.0% similarity) withN-Succinyl-L-diaminopimelic acid desuccinylase (encoded by dapE) of E.coli (7,45) (FIG. 3B) and H. influenzae (18) (39.1% identity and 58.8%similarity) (FIG. 3B). There was no substantial overall homology betweenthe products of the other complete or the two incomplete ORFs and otherknown sequences. These genes are tentatively identified as orf1, orf2,and orf3, as shown in FIG. 1.

[0086] Conservation of gidA, dapE, and orf2 Among H. pylori Strains.

[0087] To determine whether other H. pylori strains possess sequenceshomologous to gidA, dapE, or orf2, 21 strains (10 cagA⁺ and 11 cagA⁻strains) were studied by colony hybridization, using PCR-amplifiedgidA, dapE, and orf2 specific fragments. A positive signal was obtainedfrom each of these strains, indicating that these genes are conserved inH. pylori, despite other genotypic variations.

[0088] Characterization of a dapE Mutant.

[0089] To create a dapE mutant, H. pylori strain 60190 was transformedwith pMAK36 (dapE:km), and plated transformants on kanamycin-containingmedium including 1 mM DAP. Southern and PCR analysis of thekanamycin-resistant transformants indicated that the km cassette wasstably incorporated into the single chromosomal dapE gene creating adapE mutant. However, in repeated experiments, transformation of H.pylori with pMAK36 (dapE:km) on plates lacking DAP did not yield anytransformants (Table 2). Similarly, the dapE⁻ mutants obtained onDAP-containing plates were unable to grow when re-plated on TSA agar orBrucella agar with 5% FCS without the addition of DAP. Transformation ofH. pylori with pCTB8:vacA:km yielded a similar number of transformantswhether or not DAP was present in the selective media and served as apositive control. The minimum DAP concentration required for survival ofthe dapE⁻ mutant was found to be 0.2 mM (Table 3). The dapE⁻ mutant wasunable to grow on media supplemented with lysine only (Table 2),emphasizing the specific DAP requirement of H. pylori for growth and/orsurvival. TABLE 2 Growth of wild type and mutant H.pylori strains onbrucella agar in the presence or absence of DAP Medium^(a) DAP(1)DAP(+)^(b) DAP(−) Strain lysine(−) lysine(−) lysine(+)^(c) 60190 − − −60190 vacA:km + + + 60190 dapE:km − + − 60190 orf2:km + + +

[0090] TABLE 3 Minimum concentration of DAP required for growth of thedapE- H.pylori mutant DAP^(a) 2 mM 1.5 mM 1 mM 0.5 mM 0.2 mM 0.1 mM 0 mM60190 − − − − − − − 60190 dapE:km^(b) 1 + + + + + 2 + + + + + + −3 + + + + + + 60190 + + + + + + + vacA: km^(c)1 + + + + + + +2 + + + + + + + 3 + + + + + + +

[0091] Characterization of an H. pylori Mutant Lacking orf2.

[0092] The orf2 ORF begins only 80 bp downstream from dapE and lacks itsown consensus promoter, suggesting that these genes could beco-transcribed and their products could be functionally related. To testthis hypothesis, orf2 was disrupted by insertional mutagenesis, anddemonstrated the authenticity of H. pylori mutant 60190 orf2:km bySouthern hybridization and PCR. However, the orf2⁻ mutant was found togrow well with or without exogenous DAP in the growth medium (Table 2),demonstrating that orf2 is not required in the metabolic pathway leadingto DAP formation.

[0093] Evidence That Mutation of gidA is Lethal in H. pylori.

[0094] The dapE open reading frame is separated by only 10 bp from gidA,suggesting co-transcription and a functional relationship between thesetwo genes. To determine whether the gidA product in H. pylori isassociated with dapE synthesis, an attempt was made to insertionallyinactivate gidA. However, efforts to inactivate gidA by transforming H.pylori strain 60190 with pAK2:gidA:km were unsuccessful. Notransformants were observed even on media supplemented with DAP (orlysine), while parallel transformations that led to the inactivation ofdapE or vacA yielded more than 100 transformants for each. Since thesedata suggested that interruption of gidA was lethal for H. pylori, PCRwas performed to determine whether insertion of the kanamycin cassettewithin gidA had occurred to transiently create 60190 gidA:km, but thisorganism was not viable. As a positive control, wild type strain 60190was incubated in parallel with pCTB8: vacA:km to create an insertion invacA.

[0095] Agarose gel electrophoresis was performed on of PCR-amplifiedproducts from DNA isolated from wild type strain 60190 after overnightincubation and transformation with or without specified plasmid. AllPCRs used a forward primer specific for km (primer 10 in FIG. 1 andTable 1). The reverse primer was either specific for a region of the H.pylori chromosome not included in the fragment cloned in pAK2 (primer 8in FIG. 1 and Table 1) or was specific for vacA (primer 17 in Table 1)as a control. Lane 1: Template isolated from DNA strain 60190 afterovernight incubation with pAK2:gidA:km, and primers 10 and 8. Lane 2:Template DNA isolated from strain 60190, and primers 10 and 8. Lane 3:Template is a mixture of DNA from strain 60190 and pAK2:gidA:km, andprimers 10 and 8. Lane 4: Template: DNA from strain 60190 afterovernight incubation with pCTB8:vacA:km, and primers 10 and 17.

[0096] When the forward km primer (primer 10 in FIG. 1 and Table 1) andreverse vacA primer (primer 17 in Table 1) were used, a 3.1 kb band wasamplified, as expected. Using a forward km primer (primer 10) and areverse primer (primer 8 in FIG. 1 and Table 1) that is not present inpAK2 (FIG. 1), a 4.2 kb band was amplified in DNA isolated from wildtype strain 60190 that had been incubated overnight with pAK2:gidA:km.No band was present in DNA from wild type strain 60190 alone, or if60190 DNA was mixed with pAK2:gidA:km in the absence of H. pylon cells.

[0097] These results indicate that homologous recombination had occurredbetween the chromosomal DNA of the wild type strain 60190 andpAK2:gidA:km leading to km insertion within gidA, and provided furtherevidence that this transformation event was lethal to H. pylori.

[0098] RT-PCR and Slot-Blot Analysis.

[0099] To ascertain whether gidA, dapE, and orf2 are co-transcribed, RNAwas extracted for analysis from wild type H. pylori strain 60190 and itsdapE⁻ and orf2⁻ mutants. Reverse transcriptase-PCR (RT-PCR) of cDNAtemplate was performed with a pair of primers bridging the gidA and dapEORFs (primers 13 and 14, Table 1).

[0100] A 0.35 kb product was detected for each strain, as expected. InRT-PCR using primers bridging the dapE-orf2 ORFs (primers 15 and 16), noproduct was detected in the dapE mutant, as expected, but both the wildtype strain and the orf2 mutant showed a product of the expected size(0.4 kb). Negative-control PCR using RNA as template showed no products.As expected, the positive control cagA probe hybridized with equalintensity to the wild type strain and its dapE and orf2 mutants. ThegidA probe hybridized to RNA with similar intensity for the wild typestrain and its dapE⁻ and orf2⁻ mutants. The dapE probe hybridizedequally well to wild type and orf2⁻ mutant RNA, and less well to itsdapE⁻ mutant. The orf2 probe hybridized well to RNA in the wild typestrain, but only weakly to RNA from the dapE and orf2 mutants (FIG. 6).The results of both sets of experiments indicate that orf2 can beco-transcribed with dapE.

[0101] The results in this study suggest that the ability of H. pylorito synthesize DAP is based only on the succinylase pathway, or that itssynthesis via the dehydrogenase and/or acetylase pathways is too low toallow for survival.

[0102] The gidA, dapE, and orf2 ORFs are closely spaced and oppositelyoriented to the flanking genes (FIG. 1), suggesting that they form anoperon. A sequence bearing strong homology to the σ⁷⁰ promoter ispresent 5′ to gidA, but no promoter-like elements were observed upstreamof dapE and orf2. The presence of a strong putative transcriptionalterminator downstream of orf2 also is consistent with the notion thatthese 3 genes form an operon, and RT-PCR and slot blot data indicatethat dapE and orf2 may be co-transcribed. The presence of anotherputative transcriptional terminator, an 80-nucleotide palindromicsequence (DG=−2.9), beginning at nucleotide 3623 to 3702 in theintergenic region between dapE and orf2, may, under certain conditions,allow for transcription of gidA and dapE without orf2.

[0103] The dapE ORF is separated by only 10 bp from gidA. In E. coli,gidA lies near the origin of replication (oriC) (29); inactivation ofgidA by transposon insertion reduces the E. coli growth rate by 20% andcauses filamentation of cells in media containing glucose (41,42).However, in H. pylori, the arrangement of gidA and dapE in the sameoperon suggests that the products of these two genes could befunctionally related. However, the inability of DAP or lysinesupplementation to permit gidA mutants to survive suggests that itscritical activity does not involve the DAP/lysine pathway.

[0104] The presence of orf2 80 bp downstream from dapE, with no uniquepromoter sequence, suggests that these two genes may be co-transcribedand that their protein products may be functionally related. RT-PCR andslot-blot results support this hypothesis, since orf2 RNA was nottranscribed in the dapE⁻ mutant. That the orf2⁻ mutant strain grewnormally without exogenous DAP indicates that the orf2 product is notrequired for DAP biosynthesis.

[0105] The observations made in this study suggest that the enzymesinvolved in DAP biosynthesis represent targets for the development ofnovel agents against H. pylori (3,4,20,21). DAP biosynthetic genes alsomay be used to stabilize shuttle plasmids for use in H. pylori in theabsence of antibiotic markers (25). If H. pylori strains carryingmutations in DAP biosynthesis genes can be constructed, plasmidscarrying the respective complementing gene could be maintained. Suchplasmids may lead to the ability to stably maintain recombinant DNA inhumans for the expression of H. pylori or heterologous antigens, and mayprovide tools in the investigation of H. pylori pathogenesis as well asfor the development of new anti-H. pylori agents.

[0106] These results also suggest that co-administration of H. pyloridapE⁻ mutant strains with a DAP supplement may serve as an immunizationstrategy. After sufficient time for evoking an immune response directedat this H. pylori strain, cessation of DAP supplementation would lead toits death. Ideally, optimal timing of supplementation could result inthe establishment of long-term immunity to H. pylori or to heterologousantigens delivered by this superb mucosal colonizer.

[0107] Throughout this application various publications are referencedby numbers within parentheses. Full citations for these publications areas follows. The disclosures of these publications in their entiretiesare hereby incorporated by reference into this application in order tomore fully describe the state of the art to which this inventionpertains.

REFERENCES

[0108] 1. Akopyants, N. S., D. Kersulyte, and D. E. Berg. 1995. CagII, anew multigene locus associated with virulence in Helicobacter pylori.Gut 37:A1

[0109] 2. Barlett, A. T. M. and P. J. White. 1985. Species of bacillusthat make a vegetative peptidoglycan containing lysine lackdiaminopimelate epimerase but have diaminopimelate dehydrogenase. J.Gen. Microbiol. 131:2145-2152.

[0110] 3. Barlett, P. A. and C. K. Marlowe. 1983. Phosphonamidates astransition-state analogue inhibitors of thermolysin. Biochemistry22:4618-4624.

[0111] 4. Berges, D. A., W. E. DeWolf, Jr., G. L. Dunn, S. F. Grappel,D. J. Newman, J. J. Taggart, and C. Gilvarg. 1986. Peptides of2-aminopimelic acid: antibacterial agents that inhibit diaminopimelicacid. J. Med. Chem. 29:85-89.

[0112] 5. Blaser, M. J., G. I. Pérez-Pérez, H. Kleanthous, T. L. Cover,R. M. Peek, P. H. Chyou, G. N. Stemmermann, and A. Nomura. 1995.Infection with Helicobacter pylori strains possessing cagA associatedwith an increased risk of developing adenocarcinoma of the stomach.Cancer Res. 55:2111-2115.

[0113] 6. Blaser, M. J., G. I. Pérez-Pérez, J. Lindenbaum, D.Schneidman, G. Van Deventer, M. Marin-Sorensen, and W. M. Weinstein.1991. Association of infection due to Helicobacter pylori with specificupper gastrointestinal pathology. Rev. Infect. Dis. 13:S704-S708.

[0114] 7. Bouvier, J., C. Richaud, W. Higgins, O. Bogler, and P.Stragier. 1992. Cloning, characterization, and expression of the dapEgene of Escherichia coli. J. Bacteriol. 174:5265-5271.

[0115] 8. Bukhari, A. I. and A. L. Taylor. 1971. Genetic analysis ofdiaminopimelic acid- and lysine-requiring mutants of Escherichia coli.J. Bacteriol. 105:844-854.

[0116] 9. Burland, V., G. Plunkett, III, D. L. Daniels, and F. R.Blattner. 1993. DNA sequence and analysis of 136 kilobases of theEscherichia coli genome: organizational symmetry around the origin ofreplication. Genomics 16:551-561.

[0117] 10. Cirillo, J. D., T. R. Weisbrod, A. Banarjee, B. R. Bloom, andJacobs, Jr. 1994. Genetic determination of the meso-Diaminopimelatebiosynthetic pathway of mycobacteria. J. Bacteriol. 176:4424-4429.

[0118] 11. Covacci, A., S. Censini, M. Bugnoli, R. Petracca, D. Burroni,G. Macchia, A. Massone, E. Papini, Z. Xiang, N. Figura, and et al. 1993.Molecular characterization of the 128-kDa immunodominant antigen ofHelicobacter pyloriassociated with cytotoxicity and duodenal ulcer.Proc. Natl. Acad. Sci. U.S.A. 90:5791-5795.

[0119] 12. Cover, T. L., Y. Glupczynski, A. P. Lage, A. Burette, M. K.R. Tummuru, G. I. Pérez-Pérez, and M. J. Blaser. 1995. Serologicdetection of infection with cagA⁺ Helicobacter pylon strains. J. Clin.Microbiol. 33:1496-1500.

[0120] 13. Cover, T. L., W. Puryear, G. I. Pérez-Pérez, and M. J.Blaser. 1991. Effect of urease on HeLa cell vacuolation induced byHelicobacter pylori cytotoxin. Infect. Immun. 59:1264-1270.

[0121] 14. Cover, T. L., M. K. R. Tummuru, P. Cao, S. A. Thompson, andM. J. Blaser. 1994. Divergence of genetic sequences for the vacuolatingcytotoxin among Helicobacter pylori strains. J. Biol. Chem.269:10566-10573.

[0122] 15. Crabtree, J. E., J. D. Taylor, J. I. Wyatt, R. V. Heatley, T.M. Shallcross, D. S. Tompkins, and B. J. Rathbone. 1991. Mucosal IgArecognition of Helicobacter pylori 120 kDa protein, peptic ulceration,and gastric pathology. Lancet 338:332-335.

[0123] 16. Devereux, J., P. Haeberli, and O. Smithies. 1984. Acomprehensive set of sequence analysis programs for the VAX. NucleicAcids Res. 12:387-395.

[0124] 17. Dewey, D. and E. Work. 1952. Diaminopimelic aciddecarboxylase. Nature 169:533-534.

[0125] 18. Fleischmann, R. D., M. D. Adams, O. White, R. A. Clayton, E.F. Kirkness, A. R. Kerlavage, C. J. Bult, J. F. Tomb, B. A. Dougherty,J. M. Merrick, K. McKenney, G. Sutton, W. Fitzhugh, C. A. Fields, J. D.Gocayne, J. D. Scott, R. Shirley, L. I. Liu, A. Glodek, J. M. Kelley, J.F. Weidman, C. A. Phillips, T. Spriggs, E. Hedblom, M. D. Cotton, T. R.Utterback, M. C. Hanna, D. T. Nguyen, D. M. Saudek, R. C. Brandon, L. D.Fine, J. L. Fritchman, J. L. Fuhrmann, N. S. M. Geoghagen, C. L. Gnehm,L. A. McDonald, K. V. Small, C. M. Fraser, H, O. Smith, and J. C.Venter. 1995. Whole-genome random sequencing and assembly of Haemophilusinfluenza Rd. Science 269:469-512.

[0126] 19. Galan, J. E., K. Nakayama, and R. Curtiss, III. 1990. Cloningand characterization of the asd gene of Salmonella typhimurium: use instable maintenance of recombinant plasmids in Salmonella vaccinestrains. Gene 94:29-35.

[0127] 20. Galardy, R. E. and Z. P. Kortylewicz. 1984. Inhibition ofCarboxypeptidase A by aldehyde and ketone substrate analogues.Biochemistry 23:2083-2087.

[0128] 21. Gelb, M. H., J. P. Svaren, and R. H. Abeles. 1985. Fluoroketone inhibitors of hydrolytic enzymes. Biochemistry 24:1813-1817.

[0129] 22. Labigne-Roussel, A., J. Harel, and L. Tompkins. 1987. Genetransfer from Escherichia coli to Campylobacter species. Development ofshuttle vectors for genetic analysis of Campylobacter jejuni. J.Bacteriol. 169:5320-5323.

[0130] 23. Lin, Y., R. Myhrman, M. L. Schrag, and M. H. Gelb. 1987.Bacterial N-succinyl-L-diaminopimelic acid desuccinylase. J. Biol. Chem.1622-1627.

[0131] 24. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1989. Molecularcloning; a laboratory manual. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

[0132] 25. Nakayama, K., S. M. Kelley, and R. Curtiss, III. 1988.Construction of an asd expression-cloning vector: stable maintenance andhigh level expression of cloned genes in a salmonella vaccine strain.Bio. Technol. 6:693-697.

[0133] 26. Nomura, A., G. N. Stemrnermann, P. Chyou, I. Kato, G. I.Pérez-Pérez, and M. J. Blaser. 1991. Helicobacter pylori infection andgastric carcinoma in a population of Japanese-Americans in Hawaii. N.Engl. J. Med. 325:1132-1136.

[0134] 27. Ogasawara, N., S. Nakai, and H. Yoshiokawa. 1994. Systematicsequencing of the 180 kilobase region of the Bacillus subtilischromosome containing the replication origin. DNA Res 1:1-14.

[0135] 28. Ogasawara, N. and H. Yoshikawa. 1992. Genes and theirorganization in the replication origin region of the bacterialchromosome. Mol. Microbiol. 6:629-634.

[0136] 29. Ogawa, T. and T. Okazaki. 1994. Cell cycle-dependenttranscription from the gid and mioC promoters of Escherichia coli. J.Bacteriol. 176:1609-1615.

[0137] 30. Parsonnet, J., G. D. Friedman, D. P. Vandersteen, Y. Chang,J. H. Vogelman, N. Orentreich, and R. K. Sibley. 1991. Helicobacterpylori infection and the risk of gastric carcinoma. N. Engl. J Med325:1127-1131.

[0138] 31. Patte, J. C. 1996. Biosynthesis of Threonine and lysine, p.532-535. In F. C. Neidhardt (ed.), Escherichia coli and Salmonella,cellular and molecular biology. ASM Press, Washington, D.C.

[0139] 32. Peek, R. M., Jr., S. A. Thompson, J. C. Atherton, M. J.Blaser, and G. G. Miller. 1996. Expression of a novel ulcer-associatedH. pylori gene, iceA, after contact with gastric epithelium.Gastroenterol. (In Press)

[0140] 33. Rosenberg, M. and D. Court. 1979. Regulatory sequencesinvolved in the promotion and termination of RNA transcription. ( )13:319-353.

[0141] 34. Schrumpf, B., A. Schwarzer, J. Kalinowski, A. Puhler, L.Eggeling, and H. Sahm. 1991. A functionally split pathway for lysinesynthesis in Corynebacterium glutamicum. J. Bacteriol. 173:4510-4516.

[0142] 35. Stragier, P., O. Danos, and J. C. Patte. 1983. Regulation ofdiaminopimelate decarboxylase synthesis in Escherichia coli III.Nucleotide sequence of the lysA gene and its regulatory region. J. Mol.Biol. 168:321-331.

[0143] 36. Sugimoto, K., A. Oka, H. Sugisaki, M. Takanami, A. Nishimura,Y. Yasuda, and Y. Hirota. 1979. Nucleotide sequence of Escherichia coliK-12 replication origin. Proc. Natl. Acad. Sci. U.S.A. 76:575-579.

[0144] 37. Talley, N. J., A. R. Zinsmeister, E. P. Dimagno, A. Weaver,H. A. Carpenter, G. I. Pérez-Pérez, and M. J. Blaser. 1991. Gastricadenocarcinoma and Helicobacter pylori infection. J. Nat. Cancer Instit.83:1734-1739.

[0145] 38. Tayor, L. A. and R. E. Rose. 1988. A correction in thenucleotide sequence of Tn 903 kanamycin resistance determinant in pUC4K.Nucleic Acids Res. 16:358-368.

[0146] 39. Tummuru, M. K. R., T. L. Cover, and M. J. Blaser. 1993.Cloning and expression of a high molecular weight major antigen ofHelicobacter pylori: evidence of linkage to cytotoxin production.Infect. Immun. 61:1799-1809.

[0147] 40. Tummuru, M. K. R., S. A. Sharma, and M. J. Blaser. 1995.Helicobacter pylori picB, a homolog of the Bordetella pertussis toxinsecretion protein, is required for induction of IL-8 in gastricepithelial cells. Mol. Microbiol. 18:867-876.

[0148] 41. von Meyenburg, K. and F. G. Hansen. 1980. The origin orreplication, oriC, of the Escherichia coli chromosome: genes near oriCand construction of oriC deletion mutations. Mol. Cell. Biol.19:137-159.

[0149] 42. von Meyenburg, K., B. B. Jorgensen, J. Nielsen, and F. G.Hansen. 1982. Promoters of the atp operon coding for the membrane-boundATP synthase Escherichia coli mapped by Tn10 insertion mutations. Mol.Gen. Genet. 188:240-248.

[0150] 43. Walker, J. E., N. J. Gay, M. Saraste, and A. N. Eberle. 1984.DNA sequence around the Escherichia coli unc operon. Completion of thesequence of a 17 kilobase segment containing asnA, oriC, unc, glms, andphoS. Biochem. J. 224:799-815.

[0151] 44. Weinberger, S. and C. Gilvarg. 1970. Bacterial distributionof the use of succinyl and acetyl blocking groups in diaminopimelic acidbiosynthesis. J. Bacteriol. 101:323-324.

[0152] 45. Wu, B., C. Georgopoulos, and D. Ang. 1992. The essentialEscherichia coli msgB gene, a multicopy suppressor of atemperature-sensitive allele of the heat shock gene grpE, is identicalto dapE. J. Bacteriol. 174:5258-5264.

[0153] 46. McKnight, S. L. and R. Kingsbury (1982) Transcriptionalcontrol signals of a eukaryotic protein-coding gene. Science 217:316.

[0154] 47. Kunkel, T. A. (1985) Rapid and efficient site-specificmutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. 82:488(1985) Rapid and efficient site-specific mutagenesis without phenotypicselection. Proc. Natl. Acad. Sci. 82:488

[0155] 48. Eaton, J. A., C. L. Brooks, D. R. Morgan, and S. Krawowka(1991). Essential role of urease in pathogenesis of gastritis induced byHelicobacter pylori in gnotobiotic piglets. Infect. Immun. 59:2470-5.

[0156] 49. Eaton, K. A., D. R. Morgan and S. Krakowka (1992) Motility asa factor in the colonisation of gnotobiotic piglets by Helicobacterpylori. J. Med. Microbiol. 37:123-7.

1 30 1 1866 DNA Artificial Sequence CDS (1)...(1863) Description ofArtificial Sequence/ Note = synthetic construct 1 gtg gta aaa gaa agtgat att tta gtg att ggt ggg ggg cat gcg ggc 48 Val Val Lys Glu Ser AspIle Leu Val Ile Gly Gly Gly His Ala Gly 1 5 10 15 att gaa gcg agc ttgatt gca gcc aaa atg ggg gct agg gtg cat tta 96 Ile Glu Ala Ser Leu IleAla Ala Lys Met Gly Ala Arg Val His Leu 20 25 30 atc acc atg ctc ata gacacg atc ggt tta gcg agc tgt aac ccg gcg 144 Ile Thr Met Leu Ile Asp ThrIle Gly Leu Ala Ser Cys Asn Pro Ala 35 40 45 att ggg ggc ttg ggt aaa gggcat ttg act aaa gaa gtg gat gtt tta 192 Ile Gly Gly Leu Gly Lys Gly HisLeu Thr Lys Glu Val Asp Val Leu 50 55 60 ggg ggg gct atg ggg att att acagat cat agc ggt ttg caa tat cgt 240 Gly Gly Ala Met Gly Ile Ile Thr AspHis Ser Gly Leu Gln Tyr Arg 65 70 75 80 gtg tta aac gct tct aaa ggg ccggcg gtt agg ggg act aga gcg caa 288 Val Leu Asn Ala Ser Lys Gly Pro AlaVal Arg Gly Thr Arg Ala Gln 85 90 95 att gat atg gat act tac cgc att tttgca aga aat ctt gtt tta aac 336 Ile Asp Met Asp Thr Tyr Arg Ile Phe AlaArg Asn Leu Val Leu Asn 100 105 110 acc cct aat ttg agc gtc tct caa gaaatg acc gaa agt tta atc ctt 384 Thr Pro Asn Leu Ser Val Ser Gln Glu MetThr Glu Ser Leu Ile Leu 115 120 125 gaa aac gat gag gta gtg ggc gta accacg aac att aat aac act tac 432 Glu Asn Asp Glu Val Val Gly Val Thr ThrAsn Ile Asn Asn Thr Tyr 130 135 140 aga gct aaa aaa gtg atc atc acc acaggc act ttt tta aaa ggg gtg 480 Arg Ala Lys Lys Val Ile Ile Thr Thr GlyThr Phe Leu Lys Gly Val 145 150 155 160 gtg cat att ggc gag cac caa aaccaa aac ggg cgt ttt ggg gaa aac 528 Val His Ile Gly Glu His Gln Asn GlnAsn Gly Arg Phe Gly Glu Asn 165 170 175 gct tcc aat tct tta gcc ttg aattta agg gag ctt ggc ttt aag gtg 576 Ala Ser Asn Ser Leu Ala Leu Asn LeuArg Glu Leu Gly Phe Lys Val 180 185 190 gag agg tta aaa acc ggc act tgccca aga gtg gcc ggc aat agc att 624 Glu Arg Leu Lys Thr Gly Thr Cys ProArg Val Ala Gly Asn Ser Ile 195 200 205 gat ttt gaa ggc tta gaa gag catttt ggg gat gca aac cct ccc tat 672 Asp Phe Glu Gly Leu Glu Glu His PheGly Asp Ala Asn Pro Pro Tyr 210 215 220 ttc agc tat aaa acc aaa gat tttaac ccc acc caa ctc tct tgt ttc 720 Phe Ser Tyr Lys Thr Lys Asp Phe AsnPro Thr Gln Leu Ser Cys Phe 225 230 235 240 atc act tac act aac ccc attacc cac caa atc att agg gat aat ttc 768 Ile Thr Tyr Thr Asn Pro Ile ThrHis Gln Ile Ile Arg Asp Asn Phe 245 250 255 cac cga gct ccc ctt ttt agcggt caa att gaa ggc ata ggc cca agg 816 His Arg Ala Pro Leu Phe Ser GlyGln Ile Glu Gly Ile Gly Pro Arg 260 265 270 tat tgc cct agc att gaa gataaa atc aac cgc ttt agt gaa aaa gaa 864 Tyr Cys Pro Ser Ile Glu Asp LysIle Asn Arg Phe Ser Glu Lys Glu 275 280 285 cgc cac cag ctg ttt tta gagcct caa acc att cat aaa aac gaa tat 912 Arg His Gln Leu Phe Leu Glu ProGln Thr Ile His Lys Asn Glu Tyr 290 295 300 tat atc aac ggc tta agc acctct ttg ccc cta gat gtg caa gaa aag 960 Tyr Ile Asn Gly Leu Ser Thr SerLeu Pro Leu Asp Val Gln Glu Lys 305 310 315 320 gtc att cat tct atc aaaggc tta gaa aac gcc ctc atc acg cgc tat 1008 Val Ile His Ser Ile Lys GlyLeu Glu Asn Ala Leu Ile Thr Arg Tyr 325 330 335 ggc tat gcg ata gag tatgat ttc atc cag cct aca gaa tta acc cac 1056 Gly Tyr Ala Ile Glu Tyr AspPhe Ile Gln Pro Thr Glu Leu Thr His 340 345 350 gct tta gaa acc aaa aaaatc aaa ggg ctt tat ttg gcc ggg caa atc 1104 Ala Leu Glu Thr Lys Lys IleLys Gly Leu Tyr Leu Ala Gly Gln Ile 355 360 365 aat ggg act acc ggc tatgaa gaa gcg gcg gat caa ggg ctt atg gct 1152 Asn Gly Thr Thr Gly Tyr GluGlu Ala Ala Asp Gln Gly Leu Met Ala 370 375 380 ggg att aat gcg gta ttagcc tta aag aat caa gcc ccc ttt att tta 1200 Gly Ile Asn Ala Val Leu AlaLeu Lys Asn Gln Ala Pro Phe Ile Leu 385 390 395 400 aag cgc aat gaa gcttat att ggc gtt ttg att gat gat ttg gtt act 1248 Lys Arg Asn Glu Ala TyrIle Gly Val Leu Ile Asp Asp Leu Val Thr 405 410 415 aaa ggc acg aat gagcct tac aga atg ttt act agc cga gcc gaa tac 1296 Lys Gly Thr Asn Glu ProTyr Arg Met Phe Thr Ser Arg Ala Glu Tyr 420 425 430 cgc ttg ctt tta agagag gac aac acg ctt ttt agg ttg ggc gaa cat 1344 Arg Leu Leu Leu Arg GluAsp Asn Thr Leu Phe Arg Leu Gly Glu His 435 440 445 gcc tat cgt tta gggctt atg gaa cag gat ttt tat aag gaa tta aaa 1392 Ala Tyr Arg Leu Gly LeuMet Glu Gln Asp Phe Tyr Lys Glu Leu Lys 450 455 460 aaa gat aaa caa gagata caa gac aat ctc aaa cgc ctt aaa gaa tgc 1440 Lys Asp Lys Gln Glu IleGln Asp Asn Leu Lys Arg Leu Lys Glu Cys 465 470 475 480 gtc ctt acc cctagt aaa aaa ttg tta aaa cgc ttg aac gaa tta gac 1488 Val Leu Thr Pro SerLys Lys Leu Leu Lys Arg Leu Asn Glu Leu Asp 485 490 495 gaa aac cct atcaat gac aag gtt aat ggc gtt agt ttg tta gca cgc 1536 Glu Asn Pro Ile AsnAsp Lys Val Asn Gly Val Ser Leu Leu Ala Arg 500 505 510 gat agt ttt aatgca gaa aaa atg cgc tcc ttt ttc agc ttt tta gcc 1584 Asp Ser Phe Asn AlaGlu Lys Met Arg Ser Phe Phe Ser Phe Leu Ala 515 520 525 ccc ttg aac gagcgg gtt tta gag cag att aaa att gaa tgc aaa tat 1632 Pro Leu Asn Glu ArgVal Leu Glu Gln Ile Lys Ile Glu Cys Lys Tyr 530 535 540 aat att tat attgaa aag caa cac gaa aat atc gct aaa atg gat agc 1680 Asn Ile Tyr Ile GluLys Gln His Glu Asn Ile Ala Lys Met Asp Ser 545 550 555 560 atg ctc aaagtt tct atc cct aaa ggt ttt gtg ttt aaa ggc att cca 1728 Met Leu Lys ValSer Ile Pro Lys Gly Phe Val Phe Lys Gly Ile Pro 565 570 575 ggc tta agctta gaa gcg gta gaa aaa tta gaa aaa ttc cgc ccc aaa 1776 Gly Leu Ser LeuGlu Ala Val Glu Lys Leu Glu Lys Phe Arg Pro Lys 580 585 590 agc ctt tttgaa gcc tca gaa atc agc ggg atc act cca gcg aat tta 1824 Ser Leu Phe GluAla Ser Glu Ile Ser Gly Ile Thr Pro Ala Asn Leu 595 600 605 gac gtt ttgcat tta tac atc cat ttg cga aaa aac tct taa 1866 Asp Val Leu His Leu TyrIle His Leu Arg Lys Asn Ser 610 615 620 2 621 PRT Artificial SequenceDescription of Artificial Sequence\Note = synthetic construct 2 Val ValLys Glu Ser Asp Ile Leu Val Ile Gly Gly Gly His Ala Gly 1 5 10 15 IleGlu Ala Ser Leu Ile Ala Ala Lys Met Gly Ala Arg Val His Leu 20 25 30 IleThr Met Leu Ile Asp Thr Ile Gly Leu Ala Ser Cys Asn Pro Ala 35 40 45 IleGly Gly Leu Gly Lys Gly His Leu Thr Lys Glu Val Asp Val Leu 50 55 60 GlyGly Ala Met Gly Ile Ile Thr Asp His Ser Gly Leu Gln Tyr Arg 65 70 75 80Val Leu Asn Ala Ser Lys Gly Pro Ala Val Arg Gly Thr Arg Ala Gln 85 90 95Ile Asp Met Asp Thr Tyr Arg Ile Phe Ala Arg Asn Leu Val Leu Asn 100 105110 Thr Pro Asn Leu Ser Val Ser Gln Glu Met Thr Glu Ser Leu Ile Leu 115120 125 Glu Asn Asp Glu Val Val Gly Val Thr Thr Asn Ile Asn Asn Thr Tyr130 135 140 Arg Ala Lys Lys Val Ile Ile Thr Thr Gly Thr Phe Leu Lys GlyVal 145 150 155 160 Val His Ile Gly Glu His Gln Asn Gln Asn Gly Arg PheGly Glu Asn 165 170 175 Ala Ser Asn Ser Leu Ala Leu Asn Leu Arg Glu LeuGly Phe Lys Val 180 185 190 Glu Arg Leu Lys Thr Gly Thr Cys Pro Arg ValAla Gly Asn Ser Ile 195 200 205 Asp Phe Glu Gly Leu Glu Glu His Phe GlyAsp Ala Asn Pro Pro Tyr 210 215 220 Phe Ser Tyr Lys Thr Lys Asp Phe AsnPro Thr Gln Leu Ser Cys Phe 225 230 235 240 Ile Thr Tyr Thr Asn Pro IleThr His Gln Ile Ile Arg Asp Asn Phe 245 250 255 His Arg Ala Pro Leu PheSer Gly Gln Ile Glu Gly Ile Gly Pro Arg 260 265 270 Tyr Cys Pro Ser IleGlu Asp Lys Ile Asn Arg Phe Ser Glu Lys Glu 275 280 285 Arg His Gln LeuPhe Leu Glu Pro Gln Thr Ile His Lys Asn Glu Tyr 290 295 300 Tyr Ile AsnGly Leu Ser Thr Ser Leu Pro Leu Asp Val Gln Glu Lys 305 310 315 320 ValIle His Ser Ile Lys Gly Leu Glu Asn Ala Leu Ile Thr Arg Tyr 325 330 335Gly Tyr Ala Ile Glu Tyr Asp Phe Ile Gln Pro Thr Glu Leu Thr His 340 345350 Ala Leu Glu Thr Lys Lys Ile Lys Gly Leu Tyr Leu Ala Gly Gln Ile 355360 365 Asn Gly Thr Thr Gly Tyr Glu Glu Ala Ala Asp Gln Gly Leu Met Ala370 375 380 Gly Ile Asn Ala Val Leu Ala Leu Lys Asn Gln Ala Pro Phe IleLeu 385 390 395 400 Lys Arg Asn Glu Ala Tyr Ile Gly Val Leu Ile Asp AspLeu Val Thr 405 410 415 Lys Gly Thr Asn Glu Pro Tyr Arg Met Phe Thr SerArg Ala Glu Tyr 420 425 430 Arg Leu Leu Leu Arg Glu Asp Asn Thr Leu PheArg Leu Gly Glu His 435 440 445 Ala Tyr Arg Leu Gly Leu Met Glu Gln AspPhe Tyr Lys Glu Leu Lys 450 455 460 Lys Asp Lys Gln Glu Ile Gln Asp AsnLeu Lys Arg Leu Lys Glu Cys 465 470 475 480 Val Leu Thr Pro Ser Lys LysLeu Leu Lys Arg Leu Asn Glu Leu Asp 485 490 495 Glu Asn Pro Ile Asn AspLys Val Asn Gly Val Ser Leu Leu Ala Arg 500 505 510 Asp Ser Phe Asn AlaGlu Lys Met Arg Ser Phe Phe Ser Phe Leu Ala 515 520 525 Pro Leu Asn GluArg Val Leu Glu Gln Ile Lys Ile Glu Cys Lys Tyr 530 535 540 Asn Ile TyrIle Glu Lys Gln His Glu Asn Ile Ala Lys Met Asp Ser 545 550 555 560 MetLeu Lys Val Ser Ile Pro Lys Gly Phe Val Phe Lys Gly Ile Pro 565 570 575Gly Leu Ser Leu Glu Ala Val Glu Lys Leu Glu Lys Phe Arg Pro Lys 580 585590 Ser Leu Phe Glu Ala Ser Glu Ile Ser Gly Ile Thr Pro Ala Asn Leu 595600 605 Asp Val Leu His Leu Tyr Ile His Leu Arg Lys Asn Ser 610 615 6203 1167 DNA Artificial Sequence CDS (1)...(1164) Description ofArtificial Sequence\Note = synthetic construct 3 atg aac gct tta gaa atcacc caa aag ctc atc agc tac ccc acc att 48 Met Asn Ala Leu Glu Ile ThrGln Lys Leu Ile Ser Tyr Pro Thr Ile 1 5 10 15 acg ccc aaa gaa tgc ggtatt ttt gaa tac att aaa tcg ctt ttt cct 96 Thr Pro Lys Glu Cys Gly IlePhe Glu Tyr Ile Lys Ser Leu Phe Pro 20 25 30 gct ttt aaa aca cta gag tgtgga gaa aat ggc gtg aaa aac ctt ttt 144 Ala Phe Lys Thr Leu Glu Cys GlyGlu Asn Gly Val Lys Asn Leu Phe 35 40 45 tta tac cgc att ttt aac ccc cccaaa gag cat gca gaa aaa gaa cat 192 Leu Tyr Arg Ile Phe Asn Pro Pro LysGlu His Ala Glu Lys Glu His 50 55 60 gca aaa gaa aag cat gca aaa gaa aatgtt aag ccc ttg cat ttt tct 240 Ala Lys Glu Lys His Ala Lys Glu Asn ValLys Pro Leu His Phe Ser 65 70 75 80 ttt gca ggg cat att gat gtc gtg cctcct gga gat aat tgg caa agc 288 Phe Ala Gly His Ile Asp Val Val Pro ProGly Asp Asn Trp Gln Ser 85 90 95 gat ccc ttt aaa ccc atc att aaa gag gggttt tta tac ggc cgt ggg 336 Asp Pro Phe Lys Pro Ile Ile Lys Glu Gly PheLeu Tyr Gly Arg Gly 100 105 110 gcg caa gac atg aaa ggg ggc gtg ggg gcgttt ttg agc gcg agt tta 384 Ala Gln Asp Met Lys Gly Gly Val Gly Ala PheLeu Ser Ala Ser Leu 115 120 125 aat ttt aac cct aaa acc cct ttt ttg ctttct att tta ctc acg agc 432 Asn Phe Asn Pro Lys Thr Pro Phe Leu Leu SerIle Leu Leu Thr Ser 130 135 140 gat gaa gaa ggg cca ggg att ttt ggc acaaaa ctc atg cta gaa aaa 480 Asp Glu Glu Gly Pro Gly Ile Phe Gly Thr LysLeu Met Leu Glu Lys 145 150 155 160 ctc aaa gaa aaa gat tta ttg ccc catatg gcg att gtg gct gaa ccc 528 Leu Lys Glu Lys Asp Leu Leu Pro His MetAla Ile Val Ala Glu Pro 165 170 175 act tgc gaa aaa gtc tta ggc gat agcatc aaa att ggt cga aga ggt 576 Thr Cys Glu Lys Val Leu Gly Asp Ser IleLys Ile Gly Arg Arg Gly 180 185 190 tcc att aat ggc aga ctc att tta aaaggc gtt caa ggg cat gtg gct 624 Ser Ile Asn Gly Arg Leu Ile Leu Lys GlyVal Gln Gly His Val Ala 195 200 205 tac cca caa aaa tgc caa aac ccc attgat acg ctc gct tct gtt ttg 672 Tyr Pro Gln Lys Cys Gln Asn Pro Ile AspThr Leu Ala Ser Val Leu 210 215 220 cct tca att tca gga gtc cat tta gacgat ggc gat gaa tat ttt gac 720 Pro Ser Ile Ser Gly Val His Leu Asp AspGly Asp Glu Tyr Phe Asp 225 230 235 240 cct tca aaa ttg gtt gtc acc aacttg cat gca ggg tta ggg gct aat 768 Pro Ser Lys Leu Val Val Thr Asn LeuHis Ala Gly Leu Gly Ala Asn 245 250 255 aat gtg act cca ggg agc gta gaaatt acc ttt aat gcg cgc cat tct 816 Asn Val Thr Pro Gly Ser Val Glu IleThr Phe Asn Ala Arg His Ser 260 265 270 tta aaa acc acc aaa gag agt ttgaaa gaa tat tta gaa aaa gtt tta 864 Leu Lys Thr Thr Lys Glu Ser Leu LysGlu Tyr Leu Glu Lys Val Leu 275 280 285 aaa gat ttg cct cac act tta gaatta gag tca agc agt tcg cct ttc 912 Lys Asp Leu Pro His Thr Leu Glu LeuGlu Ser Ser Ser Ser Pro Phe 290 295 300 atc acg gct tct cat tca aag cttacc agc gtt tta aaa gaa aat att 960 Ile Thr Ala Ser His Ser Lys Leu ThrSer Val Leu Lys Glu Asn Ile 305 310 315 320 tta aaa aca tgc cgc acc accccc ctt tta aac acc aaa ggc ggc acg 1008 Leu Lys Thr Cys Arg Thr Thr ProLeu Leu Asn Thr Lys Gly Gly Thr 325 330 335 agc gat gcg cga ttt ttt agcgct cat ggt ata gaa gtg gtg gag ttt 1056 Ser Asp Ala Arg Phe Phe Ser AlaHis Gly Ile Glu Val Val Glu Phe 340 345 350 ggc gtt att aat gac agg atccat gcc att gat gaa agg gtg agc ttg 1104 Gly Val Ile Asn Asp Arg Ile HisAla Ile Asp Glu Arg Val Ser Leu 355 360 365 aaa gaa tta gag ctt tta gaaaaa gtg ttt ttg ggg gtt tta gag ggc 1152 Lys Glu Leu Glu Leu Leu Glu LysVal Phe Leu Gly Val Leu Glu Gly 370 375 380 ttg agt gag gca taa 1167 LeuSer Glu Ala 385 4 388 PRT Artificial Sequence Description of ArtificialSequence\Note = synthetic construct 4 Met Asn Ala Leu Glu Ile Thr GlnLys Leu Ile Ser Tyr Pro Thr Ile 1 5 10 15 Thr Pro Lys Glu Cys Gly IlePhe Glu Tyr Ile Lys Ser Leu Phe Pro 20 25 30 Ala Phe Lys Thr Leu Glu CysGly Glu Asn Gly Val Lys Asn Leu Phe 35 40 45 Leu Tyr Arg Ile Phe Asn ProPro Lys Glu His Ala Glu Lys Glu His 50 55 60 Ala Lys Glu Lys His Ala LysGlu Asn Val Lys Pro Leu His Phe Ser 65 70 75 80 Phe Ala Gly His Ile AspVal Val Pro Pro Gly Asp Asn Trp Gln Ser 85 90 95 Asp Pro Phe Lys Pro IleIle Lys Glu Gly Phe Leu Tyr Gly Arg Gly 100 105 110 Ala Gln Asp Met LysGly Gly Val Gly Ala Phe Leu Ser Ala Ser Leu 115 120 125 Asn Phe Asn ProLys Thr Pro Phe Leu Leu Ser Ile Leu Leu Thr Ser 130 135 140 Asp Glu GluGly Pro Gly Ile Phe Gly Thr Lys Leu Met Leu Glu Lys 145 150 155 160 LeuLys Glu Lys Asp Leu Leu Pro His Met Ala Ile Val Ala Glu Pro 165 170 175Thr Cys Glu Lys Val Leu Gly Asp Ser Ile Lys Ile Gly Arg Arg Gly 180 185190 Ser Ile Asn Gly Arg Leu Ile Leu Lys Gly Val Gln Gly His Val Ala 195200 205 Tyr Pro Gln Lys Cys Gln Asn Pro Ile Asp Thr Leu Ala Ser Val Leu210 215 220 Pro Ser Ile Ser Gly Val His Leu Asp Asp Gly Asp Glu Tyr PheAsp 225 230 235 240 Pro Ser Lys Leu Val Val Thr Asn Leu His Ala Gly LeuGly Ala Asn 245 250 255 Asn Val Thr Pro Gly Ser Val Glu Ile Thr Phe AsnAla Arg His Ser 260 265 270 Leu Lys Thr Thr Lys Glu Ser Leu Lys Glu TyrLeu Glu Lys Val Leu 275 280 285 Lys Asp Leu Pro His Thr Leu Glu Leu GluSer Ser Ser Ser Pro Phe 290 295 300 Ile Thr Ala Ser His Ser Lys Leu ThrSer Val Leu Lys Glu Asn Ile 305 310 315 320 Leu Lys Thr Cys Arg Thr ThrPro Leu Leu Asn Thr Lys Gly Gly Thr 325 330 335 Ser Asp Ala Arg Phe PheSer Ala His Gly Ile Glu Val Val Glu Phe 340 345 350 Gly Val Ile Asn AspArg Ile His Ala Ile Asp Glu Arg Val Ser Leu 355 360 365 Lys Glu Leu GluLeu Leu Glu Lys Val Phe Leu Gly Val Leu Glu Gly 370 375 380 Leu Ser GluAla 385 5 753 DNA Artificial Sequence CDS (1)...(750) Description ofArtificial Sequence\Note = synthetic construct 5 atg cta gga agc gtt aaaaaa acc ttt ttt tgg gtc ttg tgt ttg ggc 48 Met Leu Gly Ser Val Lys LysThr Phe Phe Trp Val Leu Cys Leu Gly 1 5 10 15 gcg ttg tgt tta aga gggtta atg gca gag cca gac gct aaa gag ctt 96 Ala Leu Cys Leu Arg Gly LeuMet Ala Glu Pro Asp Ala Lys Glu Leu 20 25 30 gtt aat tta ggc ata gag agcgcg aag aag caa gat ttc gct caa gct 144 Val Asn Leu Gly Ile Glu Ser AlaLys Lys Gln Asp Phe Ala Gln Ala 35 40 45 aaa acg cat ttt gaa aaa gct tgtgag tta aaa aat ggc ttt ggg tgt 192 Lys Thr His Phe Glu Lys Ala Cys GluLeu Lys Asn Gly Phe Gly Cys 50 55 60 gtt ttt tta ggg gcg ttc tat gaa gaaggg aaa gga gtg gga aaa gac 240 Val Phe Leu Gly Ala Phe Tyr Glu Glu GlyLys Gly Val Gly Lys Asp 65 70 75 80 ttg aaa aaa gcc atc cag ttt tac actaaa agt tgt gaa tta aat gat 288 Leu Lys Lys Ala Ile Gln Phe Tyr Thr LysSer Cys Glu Leu Asn Asp 85 90 95 ggt tat ggg tgc aac ctg cta gga aat ttatac tat aac gga caa ggc 336 Gly Tyr Gly Cys Asn Leu Leu Gly Asn Leu TyrTyr Asn Gly Gln Gly 100 105 110 gta tct aaa gac gct aaa aaa gcc tca caatac tac tct aaa gct tgc 384 Val Ser Lys Asp Ala Lys Lys Ala Ser Gln TyrTyr Ser Lys Ala Cys 115 120 125 gac tta aac cat gct gaa ggg tgt atg gtatta gga agc tta cac cat 432 Asp Leu Asn His Ala Glu Gly Cys Met Val LeuGly Ser Leu His His 130 135 140 tat ggc gta ggc acg cct aag gat tta agaaag gct ctt gat ttg tat 480 Tyr Gly Val Gly Thr Pro Lys Asp Leu Arg LysAla Leu Asp Leu Tyr 145 150 155 160 gaa aaa gct tgc gat tta aaa gac agccca ggg tgt att aat gca gga 528 Glu Lys Ala Cys Asp Leu Lys Asp Ser ProGly Cys Ile Asn Ala Gly 165 170 175 tat ata tat agt gta aca aag aat tttaag gag gct atc gtt cgt tat 576 Tyr Ile Tyr Ser Val Thr Lys Asn Phe LysGlu Ala Ile Val Arg Tyr 180 185 190 tct caa gca tgc gag ttg aac gat ggtagg ggg tgt tat aat tta ggg 624 Ser Gln Ala Cys Glu Leu Asn Asp Gly ArgGly Cys Tyr Asn Leu Gly 195 200 205 gtt atg caa tac aac gct caa ggc acagca aaa gac gaa aag caa gcg 672 Val Met Gln Tyr Asn Ala Gln Gly Thr AlaLys Asp Glu Lys Gln Ala 210 215 220 gta gaa aac ttt aaa aaa ggt tgc aaatca ggc gtt aaa gaa gca tgc 720 Val Glu Asn Phe Lys Lys Gly Cys Lys SerGly Val Lys Glu Ala Cys 225 230 235 240 gac gct ctc aag gaa ttg aaa atagaa ctt tag 753 Asp Ala Leu Lys Glu Leu Lys Ile Glu Leu 245 250 6 250PRT Artificial Sequence Description of Artificial Sequence\Note =synthetic construct 6 Met Leu Gly Ser Val Lys Lys Thr Phe Phe Trp ValLeu Cys Leu Gly 1 5 10 15 Ala Leu Cys Leu Arg Gly Leu Met Ala Glu ProAsp Ala Lys Glu Leu 20 25 30 Val Asn Leu Gly Ile Glu Ser Ala Lys Lys GlnAsp Phe Ala Gln Ala 35 40 45 Lys Thr His Phe Glu Lys Ala Cys Glu Leu LysAsn Gly Phe Gly Cys 50 55 60 Val Phe Leu Gly Ala Phe Tyr Glu Glu Gly LysGly Val Gly Lys Asp 65 70 75 80 Leu Lys Lys Ala Ile Gln Phe Tyr Thr LysSer Cys Glu Leu Asn Asp 85 90 95 Gly Tyr Gly Cys Asn Leu Leu Gly Asn LeuTyr Tyr Asn Gly Gln Gly 100 105 110 Val Ser Lys Asp Ala Lys Lys Ala SerGln Tyr Tyr Ser Lys Ala Cys 115 120 125 Asp Leu Asn His Ala Glu Gly CysMet Val Leu Gly Ser Leu His His 130 135 140 Tyr Gly Val Gly Thr Pro LysAsp Leu Arg Lys Ala Leu Asp Leu Tyr 145 150 155 160 Glu Lys Ala Cys AspLeu Lys Asp Ser Pro Gly Cys Ile Asn Ala Gly 165 170 175 Tyr Ile Tyr SerVal Thr Lys Asn Phe Lys Glu Ala Ile Val Arg Tyr 180 185 190 Ser Gln AlaCys Glu Leu Asn Asp Gly Arg Gly Cys Tyr Asn Leu Gly 195 200 205 Val MetGln Tyr Asn Ala Gln Gly Thr Ala Lys Asp Glu Lys Gln Ala 210 215 220 ValGlu Asn Phe Lys Lys Gly Cys Lys Ser Gly Val Lys Glu Ala Cys 225 230 235240 Asp Ala Leu Lys Glu Leu Lys Ile Glu Leu 245 250 7 5049 DNAArtificial Sequence Description of Artificial Sequence\Note = syntheticconstruct 7 aattccctat catgaaacct aaaatcaatc tcctagggct tgtgcctactagtaaataat 60 gcttaaagcg atgcgcgtgt gcaaattcca tttttgcatg cttaaagctaaaataaaccc 120 tcccataaaa agaaagataa tcggcgatgc gtaagaagag ctgacgctagcgaattgatc 180 cacgctaaag acgctaaaaa gcaccaaagg taaaagcgcg gttgcaggcaggtcaatggt 240 ttctgtcatc caccatatcc ccattaaaac agccacccca gccacaacaggcatcgcctt 300 ataatttaag gaattaagct tggggatttc ttctacaata tgaggcagttgagaattgag 360 cgcataacag ataataagcg cgattaacac tcctcctatt aaccccaacaagtgcacgat 420 cttagtgctt ttatcatcgg tgcgcgtatc ggtatgcgta ttggcatgcgaatgattttc 480 cattttattt taccctttaa aattactaac ctccatgcta caataaaacgttttcaaaac 540 taagatttta gaaaaatcat atcaaaacag gaaaaagagt ggtaaaagaaagtgatattt 600 tagtgattgg tggggggcat gcgggcattg aagcgagctt gattgcagccaaaatggggg 660 ctagggtgca tttaatcacc atgctcatag acacgatcgg tttagcgagctgtaacccgg 720 cgattggggg cttgggtaaa gggcatttga ctaaagaagt ggatgttttagggggggcta 780 tggggattat tacagatcat agcggtttgc aatatcgtgt gttaaacgcttctaaagggc 840 cggcggttag ggggactaga gcgcaaattg atatggatac ttaccgcatttttgcaagaa 900 atcttgtttt aaacacccct aatttgagcg tctctcaaga aatgaccgaaagtttaatcc 960 ttgaaaacga tgaggtagtg ggcgtaacca cgaacattaa taacacttacagagctaaaa 1020 aagtgatcat caccacaggc acttttttaa aaggggtggt gcatattggcgagcaccaaa 1080 accaaaacgg gcgttttggg gaaaacgctt ccaattcttt agccttgaatttaagggagc 1140 ttggctttaa ggtggagagg ttaaaaaccg gcacttgccc aagagtggccggcaatagca 1200 ttgattttga aggcttagaa gagcattttg gggatgcaaa ccctccctatttcagctata 1260 aaaccaaaga ttttaacccc acccaactct cttgtttcat cacttacactaaccccatta 1320 cccaccaaat cattagggat aatttccacc gagctcccct ttttagcggtcaaattgaag 1380 gcataggccc aaggtattgc cctagcattg aagataaaat caaccgctttagtgaaaaag 1440 aacgccacca gctgttttta gagcctcaaa ccattcataa aaacgaatattatatcaacg 1500 gcttaagcac ctctttgccc ctagatgtgc aagaaaaggt cattcattctatcaaaggct 1560 tagaaaacgc cctcatcacg cgctatggct atgcgataga gtatgatttcatccagccta 1620 cagaattaac ccacgcttta gaaaccaaaa aaatcaaagg gctttatttggccgggcaaa 1680 tcaatgggac taccggctat gaagaagcgg cggatcaagg gcttatggctgggattaatg 1740 cggtattagc cttaaagaat caagccccct ttattttaaa gcgcaatgaagcttatattg 1800 gcgttttgat tgatgatttg gttactaaag gcacgaatga gccttacagaatgtttacta 1860 gccgagccga ataccgcttg cttttaagag aggacaacac gctttttaggttgggcgaac 1920 atgcctatcg tttagggctt atggaacagg atttttataa ggaattaaaaaaagataaac 1980 aagagataca agacaatctc aaacgcctta aagaatgcgt ccttacccctagtaaaaaat 2040 tgttaaaacg cttgaacgaa ttagacgaaa accctatcaa tgacaaggttaatggcgtta 2100 gtttgttagc acgcgatagt tttaatgcag aaaaaatgcg ctcctttttcagctttttag 2160 cccccttgaa cgagcgggtt ttagagcaga ttaaaattga atgcaaatataatatttata 2220 ttgaaaagca acacgaaaat atcgctaaaa tggatagcat gctcaaagtttctatcccta 2280 aaggttttgt gtttaaaggc attccaggct taagcttaga agcggtagaaaaattagaaa 2340 aattccgccc caaaagcctt tttgaagcct cagaaatcag cgggatcactccagcgaatt 2400 tagacgtttt gcatttatac atccatttgc gaaaaaactc ttaaaggatttttaatgaac 2460 gctttagaaa tcacccaaaa gctcatcagc taccccacca ttacgcccaaagaatgcggt 2520 atttttgaat acattaaatc gctttttcct gcttttaaaa cactagagtgtggagaaaat 2580 ggcgtgaaaa accttttttt ataccgcatt tttaaccccc ccaaagagcatgcagaaaaa 2640 gaacatgcaa aagaaaagca tgcaaaagaa aatgttaagc ccttgcatttttcttttgca 2700 gggcatattg atgtcgtgcc tcctggagat aattggcaaa gcgatccctttaaacccatc 2760 attaaagagg ggtttttata cggccgtggg gcgcaagaca tgaaagggggcgtgggggcg 2820 tttttgagcg cgagtttaaa ttttaaccct aaaacccctt ttttgctttctattttactc 2880 acgagcgatg aagaagggcc agggattttt ggcacaaaac tcatgctagaaaaactcaaa 2940 gaaaaagatt tattgcccca tatggcgatt gtggctgaac ccacttgcgaaaaagtctta 3000 ggcgatagca tcaaaattgg tcgaagaggt tccattaatg gcagactcattttaaaaggc 3060 gttcaagggc atgtggctta cccacaaaaa tgccaaaacc ccattgatacgctcgcttct 3120 gttttgcctt caatttcagg agtccattta gacgatggcg atgaatattttgacccttca 3180 aaattggttg tcaccaactt gcatgcaggg ttaggggcta ataatgtgactccagggagc 3240 gtagaaatta cctttaatgc gcgccattct ttaaaaacca ccaaagagagtttgaaagaa 3300 tatttagaaa aagttttaaa agatttgcct cacactttag aattagagtcaagcagttcg 3360 cctttcatca cggcttctca ttcaaagctt accagcgttt taaaagaaaatattttaaaa 3420 acatgccgca ccacccccct tttaaacacc aaaggcggca cgagcgatgcgcgatttttt 3480 agcgctcatg gtatagaagt ggtggagttt ggcgttatta atgacaggatccatgccatt 3540 gatgaaaggg tgagcttgaa agaattagag cttttagaaa aagtgtttttgggggtttta 3600 gagggcttga gtgaggcata aaataaataa acattaagta aggcttatcaatatttgatt 3660 acaattataa agggttacat ttttttaata ggagatatac catgctaggaagcgttaaaa 3720 aaaccttttt ttgggtcttg tgtttgggcg cgttgtgttt aagagggttaatggcagagc 3780 cagacgctaa agagcttgtt aatttaggca tagagagcgc gaagaagcaagatttcgctc 3840 aagctaaaac gcattttgaa aaagcttgtg agttaaaaaa tggctttgggtgtgtttttt 3900 taggggcgtt ctatgaagaa gggaaaggag tgggaaaaga cttgaaaaaagccatccagt 3960 tttacactaa aagttgtgaa ttaaatgatg gttatgggtg caacctgctaggaaatttat 4020 actataacgg acaaggcgta tctaaagacg ctaaaaaagc ctcacaatactactctaaag 4080 cttgcgactt aaaccatgct gaagggtgta tggtattagg aagcttacaccattatggcg 4140 taggcacgcc taaggattta agaaaggctc ttgatttgta tgaaaaagcttgcgatttaa 4200 aagacagccc agggtgtatt aatgcaggat atatatatag tgtaacaaagaattttaagg 4260 aggctatcgt tcgttattct caagcatgcg agttgaacga tggtagggggtgttataatt 4320 taggggttat gcaatacaac gctcaaggca cagcaaaaga cgaaaagcaagcggtagaaa 4380 actttaaaaa aggttgcaaa tcaggcgtta aagaagcatg cgacgctctcaaggaattga 4440 aaatagaact ttagtttcaa taaagttaag ccaaacgccg tgtttagctggcttctacgc 4500 tttttaatat cttaatgaaa gcataaaccc tacaaactaa tcttttaatcataataaggg 4560 ttttatatcg cacccattca ttgccgtttt tagattggcg cttgaaaggtttaaagcaag 4620 tttgttcaaa cccttaaaaa gggtttttaa cccctacaac gctttcaatagcacgctatt 4680 taggcgttcg gtaaaacttt tagcgtcttt taaagcccct ttttctaaaagcttcgcccc 4740 atcataaagc aaccagataa aagcgttcaa ctgctcttta tcttcgcattttaagagttt 4800 ttggaaaatc gcatggttag ggtttaattc tagcgttttc ttgctttcaggcacgctttg 4860 acccatttga cgcataaaat tagccatcat cgcattttgg tcatcgcctattaaagccac 4920 cgctgaagtg agatgactgg aaagctctac gcctttaatc tcatctttaagattttcttc 4980 aaacgctttc attaaatctt taaactgatc ttttatctca tcaaggatttcttccaaacc 5040 aagggttaa 5049 8 18 DNA Artificial Sequence Descriptionof Artificial Sequence\Note = synthetic construct 8 caggaaaaag agtggtaa18 9 18 DNA Artificial Sequence Description of Artificial Sequence\Note= synthetic construct 9 ttaagagttt tttcgcaa 18 10 18 DNA ArtificialSequence Description of Artificial Sequence\Note = synthetic construct10 aaggatattt aatgaacg 18 11 21 DNA Artificial Sequence Description ofArtificial Sequence\Note = synthetic construct 11 gtttatttat tttatgcctca 21 12 19 DNA Artificial Sequence Description of ArtificialSequence\Note = synthetic construct 12 taatttaggc atagagagc 19 13 21 DNAArtificial Sequence Description of Artificial Sequence\Note = syntheticconstruct 13 tataacggac aaggcgtatc t 21 14 24 DNA Artificial SequenceDescription of Artificial Sequence\Note = synthetic construct 14gttctatttt caattccttg agag 24 15 18 DNA Artificial Sequence Descriptionof Artificial Sequence\Note = synthetic construct 15 gcgtgaatga atacgata18 16 24 DNA Artificial Sequence Description of Artificial Sequence\Note= synthetic construct 16 ctcccaccag cttatatacc ttag 24 17 21 DNAArtificial Sequence Description of Artificial Sequence\Note = syntheticconstruct 17 ctggggatca agcctgattg g 21 18 20 DNA Artificial SequenceDescription of Artificial Sequence\Note = synthetic construct 18gaccgttccg tggcaaagca 20 19 22 DNA Artificial Sequence Description ofArtificial Sequence\Note = synthetic construct 19 cttgtgcaat gtaacatcagag 22 20 18 DNA Artificial Sequence Description of ArtificialSequence\Note = synthetic construct 20 gcattccagg cttaagct 18 21 20 DNAArtificial Sequence Description of Artificial Sequence\Note = syntheticconstruct 21 tgcatgttct ttttctgcat 20 22 18 DNA Artificial SequenceDescription of Artificial Sequence\Note = synthetic construct 22gagtttggcg ttattaat 18 23 17 DNA Artificial Sequence Description ofArtificial Sequence\Note = synthetic construct 23 gctttttcaa aatgcgt 1724 15 DNA Artificial Sequence Description of Artificial Sequence\Note =synthetic construct 24 aagcttgatc actcc 15 25 613 PRT H. influenzae 25Met Phe Tyr Thr Glu Thr Tyr Asp Val Ile Val Ile Gly Gly Gly His 1 5 1015 Ala Gly Thr Glu Ala Ala Leu Ala Pro Ala Arg Met Gly Phe Lys Thr 20 2530 Leu Leu Leu Thr His Asn Val Asp Thr Leu Gly Gln Met Ser Cys Asn 35 4045 Pro Ala Ile Gly Gly Ile Gly Lys Gly His Leu Val Lys Glu Val Asp 50 5560 Ala Met Gly Gly Leu Met Ala His Ala Ala Asp Lys Ala Gly Ile Gln 65 7075 80 Phe Arg Thr Leu Asn Ser Ser Lys Gly Pro Ala Val Arg Ala Thr Arg 8590 95 Ala Gln Ser Asp Arg Val Leu Tyr Arg Gln Ala Val Arg Thr Ala Leu100 105 110 Glu Asn Gln Pro Asn Leu Asp Ile Phe Gln Gln Glu Ala Thr AspIle 115 120 125 Leu Ile Glu Gln Asp Arg Val Thr Gly Val Ser Thr Lys MetGly Leu 130 135 140 Thr Phe Arg Ala Lys Ser Val Ile Leu Thr Ala Gly ThrPhe Leu Ala 145 150 155 160 Gly Lys Ile His Ile Gly Leu Glu Asn Tyr GluGly Gly Arg Ala Gly 165 170 175 Asp Ser Ala Ser Val Asn Leu Ser His ArgLeu Arg Asp Leu Gly Leu 180 185 190 Arg Val Asp Arg Leu Lys Thr Gly ThrPro Pro Arg Ile Asp Ala Arg 195 200 205 Thr Ile Asn Phe Asp Ile Leu AlaLys Gln His Gly Asp Glu Val Leu 210 215 220 Pro Val Phe Ser Phe Met GlySer Val Asp Asp His Pro Gln Gln Ile 225 230 235 240 Pro Cys Tyr Ile ThrHis Thr Asn Glu Gln Thr His Glu Val Ile Arg 245 250 255 Asn Asn Leu AspArg Ser Pro Met Tyr Thr Gly Val Ile Glu Gly Ile 260 265 270 Gly Pro ArgTyr Cys Pro Ser Ile Glu Asp Lys Val Met Arg Phe Ala 275 280 285 Asp ArgAsn Ser His Gln Ile Tyr Leu Glu Pro Glu Gly Leu Thr Ser 290 295 300 AsnGlu Val Tyr Pro Asn Gly Ile Ser Thr Ser Leu Pro Phe Asp Val 305 310 315320 Gln Met Gly Ile Val Asn Ser Met Lys Gly Leu Glu Asn Ala Arg Ile 325330 335 Val Lys Pro Gly Tyr Ala Ile Glu Tyr Asp Tyr Phe Asp Pro Arg Asp340 345 350 Leu Lys Pro Thr Leu Glu Thr Lys Ser Ile Ser Gly Leu Phe PheAla 355 360 365 Gly Gln Ile Asn Gly Thr Thr Gly Tyr Glu Glu Ala Ala AlaGln Gly 370 375 380 Leu Leu Ala Gly Ile Asn Ala Gly Leu Tyr Val Gln GluLys Asp Ala 385 390 395 400 Trp Tyr Pro Arg Arg Asp Gln Ser Tyr Thr GlyVal Leu Val Asp Asp 405 410 415 Leu Cys Thr Leu Gly Thr Lys Glu Pro TyrArg Val Phe Thr Ser Arg 420 425 430 Ala Glu Tyr Arg Leu Leu Leu Arg GluAsp Asn Ala Asp Ile Arg Leu 435 440 445 Thr Pro Ile Ala His Glu Leu GlyLeu Ile Asp Glu Ala Arg Trp Ala 450 455 460 Arg Phe Asn Gln Lys Met GluAsn Ile Glu Gln Glu Arg Gln Arg Leu 465 470 475 480 Arg Ser Ile Trp LeuHis Pro Arg Ser Glu Tyr Leu Glu Glu Ala Asn 485 490 495 Lys Val Leu GlySer Pro Leu Val Arg Glu Ala Ser Gly Glu Asp Leu 500 505 510 Leu Arg ArgPro Glu Met Thr Tyr Asp Ile Leu Thr Ser Leu Thr Pro 515 520 525 Tyr LysPro Ala Met Glu Asp Lys Glu Ala Val Glu Gln Val Glu Ile 530 535 540 AlaIle Lys Tyr Gln Gly Tyr Ile Glu His Gln Gln Asn Phe Asp Tyr 545 550 555560 Ser Lys Val Ser Gly Leu Ser Asn Glu Val Arg Ala Lys Leu Glu Gln 565570 575 His Arg Pro Val Ser Ile Gly Gln Ala Ser Arg Ile Ser Gly Ile Thr580 585 590 Pro Ala Ala Ile Ser Ile Ile Leu Val Asn Leu Lys Lys Gln GlyMet 595 600 605 Leu Lys Arg Gly Glu 610 26 621 PRT H. pylori 26 Met ValLys Glu Ser Asp Ile Leu Val Ile Gly Gly Gly His Ala Gly 1 5 10 15 IleGlu Ala Ser Leu Ile Ala Ala Lys Met Gly Ala Arg Val His Leu 20 25 30 IleThr Met Leu Ile Asp Thr Ile Gly Leu Ala Ser Cys Asn Pro Ala 35 40 45 IleGly Gly Leu Gly Lys Gly His Leu Thr Lys Glu Val Asp Val Leu 50 55 60 GlyGly Ala Met Gly Ile Ile Thr Asp His Ser Gly Leu Gln Tyr Arg 65 70 75 80Val Leu Asn Ala Ser Lys Gly Pro Ala Val Arg Gly Thr Arg Ala Gln 85 90 95Ile Asp Met Asp Thr Tyr Arg Ile Phe Ala Arg Asn Leu Val Leu Asn 100 105110 Thr Pro Asn Leu Ser Val Ser Gln Glu Met Thr Glu Ser Leu Ile Leu 115120 125 Glu Asn Asp Glu Val Val Gly Val Thr Thr Asn Ile Asn Asn Thr Tyr130 135 140 Arg Ala Lys Lys Val Ile Ile Thr Thr Gly Thr Phe Leu Lys GlyVal 145 150 155 160 Val His Ile Gly Glu His Gln Asn Gln Asn Gly Arg PheGly Glu Asn 165 170 175 Ala Ser Asn Ser Leu Ala Ile Asn Leu Arg Glu LeuGly Phe Lys Val 180 185 190 Glu Arg Leu Lys Thr Gly Thr Cys Pro Arg ValAla Gly Asn Ser Ile 195 200 205 Asp Phe Glu Gly Leu Glu Glu His Phe GlyAsp Ala Asn Pro Pro Tyr 210 215 220 Phe Ser Tyr Lys Thr Lys Asp Phe AsnPro Thr Gln Leu Ser Cys Phe 225 230 235 240 Ile Thr Tyr Thr Asn Pro IleThr His Gln Ile Ile Arg Asp Asn Phe 245 250 255 His Arg Ala Pro Leu PheSer Gly Gln Ile Glu Gly Ile Gly Pro Arg 260 265 270 Tyr Cys Pro Ser IleGlu Asp Lys Ile Asn Arg Phe Ser Glu Lys Glu 275 280 285 Arg His Gln LeuPhe Leu Glu Pro Gln Thr Ile His Lys Asn Glu Tyr 290 295 300 Tyr Ile AsnGly Leu Ser Thr Ser Leu Pro Leu Asp Val Gln Glu Lys 305 310 315 320 ValIle His Ser Ile Lys Gly Leu Glu Asn Ala Leu Ile Thr Arg Tyr 325 330 335Gly Tyr Ala Ile Glu Tyr Asp Phe Ile Gln Pro Thr Glu Leu Thr His 340 345350 Ala Leu Glu Thr Lys Lys Ile Lys Gly Leu Tyr Leu Ala Gly Gln Ile 355360 365 Asn Gly Thr Thr Gly Tyr Glu Glu Ala Ala Asp Gln Gly Leu Met Ala370 375 380 Gly Ile Asn Ala Val Leu Ala Leu Lys Asn Gln Ala Pro Phe IleLeu 385 390 395 400 Lys Arg Asn Glu Ala Tyr Ile Gly Val Leu Ile Asp AspLeu Val Thr 405 410 415 Lys Gly Thr Asn Glu Pro Tyr Arg Met Phe Thr SerArg Ala Glu Tyr 420 425 430 Arg Leu Leu Leu Arg Glu Asp Asn Thr Leu PheArg Leu Gly Glu His 435 440 445 Ala Tyr Arg Leu Gly Leu Met Glu Gln AspPhe Tyr Lys Glu Leu Lys 450 455 460 Lys Asp Lys Gln Glu Ile Gln Asp AsnLeu Lys Arg Leu Lys Glu Cys 465 470 475 480 Val Leu Thr Pro Ser Lys LysLeu Leu Lys Arg Leu Asn Glu Leu Asp 485 490 495 Glu Asn Pro Ile Asn AspLys Val Asn Gly Val Ser Leu Leu Ala Arg 500 505 510 Asp Ser Phe Asn AlaGlu Lys Met Arg Ser Phe Phe Ser Phe Leu Ala 515 520 525 Pro Leu Asn GluArg Val Leu Glu Gln Ile Lys Ile Glu Cys Lys Tyr 530 535 540 Asn Ile TyrIle Glu Lys Gln His Glu Asn Ile Ala Lys Met Asp Ser 545 550 555 560 MetLeu Lys Val Ser Ile Pro Lys Gly Phe Val Phe Lys Gly Ile Pro 565 570 575Gly Leu Ser Leu Glu Ala Val Glu Lys Leu Glu Lys Phe Arg Pro Lys 580 585590 Ser Leu Phe Glu Ala Ser Glu Ile Ser Gly Ile Thr Pro Ala Asn Leu 595600 605 Asp Val Leu His Leu Tyr Ile His Leu Arg Lys Asn Ser 610 615 62027 626 PRT E. coli 27 Met Phe Tyr Pro Asp Pro Phe Asp Val Ile Ile IleGly Gly Gly His 1 5 10 15 Ala Gly Thr Glu Ala Ala Met Ala Ala Ala ArgMet Gly Gln Gln Thr 20 25 30 Leu Leu Leu Thr His Asn Ile Asp Thr Leu GlyGln Met Ser Cys Asn 35 40 45 Pro Ala Ile Gly Gly Ile Gly Lys Gly His LeuVal Lys Glu Val Asp 50 55 60 Ala Leu Gly Gly Leu Met Ala Lys Ala Ile AspGln Ala Gly Ile Gln 65 70 75 80 Phe Arg Ile Leu Asn Ala Ser Lys Gly ProAla Val Arg Ala Thr Arg 85 90 95 Ala Gln Ala Asp Arg Val Leu Tyr Arg GlnAla Val Arg Thr Ala Leu 100 105 110 Glu Asn Gln Pro Asn Leu Met Ile PheGln Gln Ala Val Glu Asp Leu 115 120 125 Ile Val Glu Asn Asp Arg Val ValGly Ala Val Thr Gln Met Gly Leu 130 135 140 Lys Phe Arg Ala Lys Ala ValVal Leu Thr Val Gly Thr Phe Leu Asp 145 150 155 160 Gly Lys Ile His IleGly Leu Asp Asn Tyr Ser Gly Gly Arg Ala Gly 165 170 175 Asp Pro Pro SerIle Pro Leu Ser Arg Arg Leu Arg Glu Leu Pro Leu 180 185 190 Arg Val GlyArg Leu Lys Thr Gly Thr Pro Pro Arg Ile Asp Ala Arg 195 200 205 Thr IleAsp Phe Ser Val Leu Ala Gln Gln His Gly Asp Asn Pro Met 210 215 220 ProVal Phe Ser Phe Met Gly Asn Ala Ser Gln His Pro Gln Gln Val 225 230 235240 Pro Cys Tyr Ile Thr His Thr Asn Glu Lys Thr His Asp Val Ile Arg 245250 255 Ser Asn Leu Asp Arg Ser Pro Met Tyr Ala Gly Val Ile Glu Gly Val260 265 270 Gly Pro Arg Tyr Cys Pro Ser Ile Glu Asp Lys Val Met Arg PheAla 275 280 285 Asp Arg Asn Gln His Gln Ile Phe Leu Glu Pro Glu Gly LeuThr Ser 290 295 300 Asn Glu Ile Tyr Pro Asn Gly Ile Ser Thr Ser Leu ProPhe Asp Val 305 310 315 320 Gln Met Gln Ile Val Arg Ser Met Gln Gly MetGlu Asn Ala Lys Ile 325 330 335 Val Arg Pro Gly Tyr Ala Ile Glu Tyr AspPhe Phe Asp Pro Arg Asp 340 345 350 Leu Lys Pro Thr Leu Glu Ser Lys PheIle Gln Gly Leu Phe Phe Ala 355 360 365 Gly Gln Ile Asn Gly Thr Thr GlyTyr Glu Glu Ala Ala Ala Gln Gly 370 375 380 Leu Leu Ala Gly Leu Asn AlaAla Arg Leu Ser Ala Asp Lys Glu Gly 385 390 395 400 Trp Ala Pro Ala ArgSer Gln Ala Tyr Leu Gly Val Leu Val Asp Asp 405 410 415 Leu Cys Thr LeuGly Thr Lys Glu Pro Tyr Arg Met Phe Thr Ser Arg 420 425 430 Ala Glu TyrArg Leu Met Leu Arg Glu Asp Asn Ala Asp Leu Arg Leu 435 440 445 Thr GluIle Gly Arg Glu Leu Gly Leu Val Asp Asp Glu Arg Trp Ala 450 455 460 ArgPhe Asn Glu Lys Leu Glu Asn Ile Glu Arg Glu Arg Gln Arg Leu 465 470 475480 Lys Ser Thr Trp Val Thr Pro Ser Ala Glu Ala Ala Ala Glu Val Asn 485490 495 Ala His Leu Thr Ala Pro Leu Ser Arg Glu Ala Ser Gly Glu Asp Leu500 505 510 Leu Arg Pro Glu Met Thr Tyr Glu Lys Leu Thr Thr Leu Thr ProPhe 515 520 525 Ala Pro Ala Leu Thr Asp Glu Gln Ala Ala Glu Gln Val GluIle Gln 530 535 540 Val Lys Tyr Glu Gly Tyr Ile Ala Arg Gln Gln Asp GluIle Glu Lys 545 550 555 560 Gln Leu Arg Asn Glu Asn Thr Leu Leu Pro AlaThr Leu Asp Tyr Arg 565 570 575 Gln Val Ser Gly Leu Ser Asn Glu Val IleAla Lys Leu Asn Asp His 580 585 590 Lys Pro Ala Ser Ile Gly Gln Ala SerArg Ile Ser Gly Val Thr Pro 595 600 605 Ala Ala Ile Ser Ile Leu Leu ValTrp Leu Lys Lys Gln Gly Met Leu 610 615 620 Arg Arg 625 28 355 PRT H.influenzae 28 Met Lys Glu Lys Val Val Ser Leu Ala Gln Asp Leu Ile ArgArg Pro 1 5 10 15 Ser Ile Ser Pro Asn Asp Glu Gly Cys Gln Gln Ile IleAla Glu Arg 20 25 30 Leu Glu Lys Leu Gly Phe Gln Ile Glu Trp Met Pro PheAsn Asp Thr 35 40 45 Leu Asn Leu Trp Ala Lys His Gly Thr Ser Glu Pro ValIle Ala Phe 50 55 60 Ala Gly His Thr Asp Val Val Pro Thr Gly Asp Glu AsnGln Trp Ser 65 70 75 80 Ser Pro Pro Phe Ser Ala Glu Ile Ile Asp Gly MetLeu Tyr Gly Arg 85 90 95 Gly Ala Ala Asp Met Lys Gly Ser Leu Ala Ala MetIle Val Ala Ala 100 105 110 Glu Glu Tyr Val Lys Ala Asn Pro Asn His LysGly Thr Ile Ala Leu 115 120 125 Leu Ile Thr Ser Asp Glu Glu Ala Thr AlaLys Asp Gly Thr Ile His 130 135 140 Val Val Glu Thr Leu Met Ala Arg AspGlu Lys Ile Thr Tyr Cys Met 145 150 155 160 Val Gly Glu Pro Ser Ser AlaLys Asn Leu Gly Asp Val Val Lys Asn 165 170 175 Gly Arg Arg Gly Ser IleThr Gly Asn Leu Tyr Ile Gln Gly Ile Gln 180 185 190 Gly His Val Ala TyrPro His Leu Ala Glu Asn Pro Ile His Lys Ala 195 200 205 Ala Leu Phe LeuGln Glu Leu Thr Thr Tyr Gln Trp Asp Lys Gly Asn 210 215 220 Glu Phe PhePro Pro Thr Ser Leu Gln Ile Ala Asn Ile His Ala Gly 225 230 235 240 ThrGly Ser Asn Asn Val Ile Pro Ala Glu Leu Tyr Ile Gln Phe Asn 245 250 255Leu Arg Tyr Cys Thr Glu Val Thr Asp Glu Ile Ile Lys Gln Lys Val 260 265270 Ala Glu Met Leu Glu Lys His Asn Leu Lys Tyr Arg Ile Glu Trp Asn 275280 285 Leu Ser Gly Lys Pro Phe Leu Thr Lys Pro Gly Lys Leu Leu Asp Ser290 295 300 Ile Thr Ser Ala Ile Glu Glu Thr Ile Gly Ile Thr Pro Lys AlaGlu 305 310 315 320 Thr Gly Gly Gly Thr Ser Asp Gly Arg Phe Ile Ala LeuMet Gly Ala 325 330 335 Glu Val Val Glu Phe Gly Pro Leu Asn Ser Thr IleHis Lys Val Asn 340 345 350 Glu Glu Glu 355 29 388 PRT H. pylori 29 MetAsn Ala Leu Glu Ile Thr Gln Lys Leu Ile Ser Tyr Pro Thr Ile 1 5 10 15Thr Pro Lys Glu Cys Gly Ile Phe Glu Tyr Ile Lys Ser Leu Phe Pro 20 25 30Ala Phe Lys Thr Leu Glu Cys Gly Glu Asn Gly Val Lys Asn Leu Phe 35 40 45Leu Tyr Arg Ile Phe Asn Pro Pro Lys Glu His Ala Glu Lys Glu His 50 55 60Ala Lys Glu Lys His Ala Lys Glu Asn Val Lys Pro Leu His Phe Ser 65 70 7580 Phe Ala Gly His Ile Asp Val Val Pro Pro Gly Asp Asn Trp Gln Ser 85 9095 Asp Pro Phe Lys Pro Ile Ile Lys Glu Gly Phe Leu Tyr Gly Arg Gly 100105 110 Ala Gln Asp Met Lys Gly Gly Val Gly Ala Phe Leu Ser Ala Ser Leu115 120 125 Asn Phe Asn Pro Lys Thr Pro Phe Leu Leu Ser Ile Leu Leu ThrSer 130 135 140 Asp Glu Glu Gly Pro Gly Ile Phe Gly Thr Lys Leu Met LeuGlu Lys 145 150 155 160 Leu Lys Glu Lys Asp Leu Leu Pro His Met Ala IleVal Ala Glu Pro 165 170 175 Thr Cys Glu Lys Val Leu Gly Asp Ser Ile LysIle Gly Arg Arg Gly 180 185 190 Ser Ile Asn Gly Arg Leu Ile Leu Lys GlyVal Gln Gly His Val Ala 195 200 205 Tyr Pro Gln Lys Cys Gln Asn Pro IleAsp Thr Leu Ala Ser Val Leu 210 215 220 Pro Ser Ile Ser Gly Val His LeuAsp Asp Gly Asp Glu Tyr Phe Asp 225 230 235 240 Pro Ser Lys Leu Val ValThr Asn Leu His Ala Gly Leu Gly Ala Asn 245 250 255 Asn Val Thr Pro GlySer Val Glu Ile Thr Phe Asn Ala Arg His Ser 260 265 270 Leu Lys Thr ThrLys Glu Ser Leu Lys Glu Tyr Leu Glu Lys Val Leu 275 280 285 Lys Asp LeuPro His Thr Leu Glu Leu Glu Ser Ser Ser Ser Pro Phe 290 295 300 Ile ThrAla Ser His Ser Lys Leu Thr Ser Val Leu Lys Glu Asn Ile 305 310 315 320Leu Lys Thr Cys Arg Thr Thr Pro Leu Leu Asn Thr Lys Gly Gly Thr 325 330335 Ser Asp Ala Arg Phe Phe Ser Ala His Gly Ile Glu Val Val Glu Phe 340345 350 Gly Val Ile Asn Asp Arg Ile His Ala Ile Asp Glu Arg Val Ser Leu355 360 365 Lys Glu Leu Glu Leu Leu Glu Lys Val Phe Leu Gly Val Leu GluGly 370 375 380 Leu Ser Glu Ala 385 30 370 PRT E. coli 30 Met Ser CysPro Val Ile Glu Leu Thr Gln Gln Leu Ile Arg Arg Pro 1 5 10 15 Ser LeuSer Pro Asp Asp Ala Gly Cys Gln Ala Leu Leu Ile Glu Arg 20 25 30 Leu GlnAla Ile Gly Phe Thr Val Glu Arg Met Asp Phe Ala Asp Thr 35 40 45 Gln AsnPhe Trp Ala Trp Arg Gly Gln Gly Glu Thr Leu Ala Phe Ala 50 55 60 Gly HisThr Asp Val Val Pro Pro Gly Asp Ala Asp Arg Trp Ile Asn 65 70 75 80 ProPro Phe Glu Pro Thr Ile Arg Asp Gly Met Leu Phe Gly Arg Gly 85 90 95 AlaAla Asp Met Lys Gly Ser Leu Ala Ala Met Val Val Ala Ala Glu 100 105 110Arg Phe Val Ala Gln His Pro Asn His Thr Gly Arg Leu Ala Phe Leu 115 120125 Ile Thr Ser Asp Glu Glu Ala Ser Ala His Asn Gly Thr Val Lys Val 130135 140 Val Glu Ala Leu Met Ala Arg Asn Glu Arg Leu Asp Tyr Cys Leu Val145 150 155 160 Gly Glu Pro Ser Ser Ile Glu Val Val Gly Asp Val Val LysAsn Gly 165 170 175 Arg Arg Gly Ser Leu Thr Cys Asn Leu Thr Ile His GlyVal Gln Gly 180 185 190 His Val Ala Tyr Pro His Leu Ala Asp Asn Pro ValHis Arg Ala Ala 195 200 205 Pro Phe Leu Asn Glu Leu Val Ala Ile Glu TrpAsp Gln Gly Asn Glu 210 215 220 Phe Phe Pro Ala Thr Ser Met Gln Ile AlaAsn Ile Gln Ala Gly Thr 225 230 235 240 Gly Ser Asn Asn Val Ile Pro GlyGlu Leu Phe Val Gln Phe Asn Phe 245 250 255 Arg Phe Ser Thr Glu Leu ThrAsp Glu Met Ile Lys Ala Gln Val Leu 260 265 270 Ala Leu Leu Glu Lys HisGln Leu Arg Tyr Thr Val Asp Trp Trp Leu 275 280 285 Ser Gly Gln Pro PheLeu Thr Ala Arg Gly Lys Leu Val Asp Ala Val 290 295 300 Val Asn Ala ValGlu His Tyr Asn Glu Ile Lys Pro Gln Leu Leu Thr 305 310 315 320 Thr GlyGly Thr Ser Asp Gly Arg Phe Ile Ala Arg Met Gly Ala Gln 325 330 335 ValVal Glu Leu Gly Pro Val Asn Ala Thr Ile His Lys Ile Asn Glu 340 345 350Cys Val Asn Ala Ala Asp Leu Gln Leu Gln Arg Ile Met Glu Gln Leu 355 360365 Val Ala 370

What is claimed is:
 1. An isolated dapE gene of Helicobacter pylori. 2.The dapE gene of claim 1, consisting of the nucleotide sequence definedin SEQ ID NO:1.
 3. A Helicobacter pylori-specific nucleic acid fragmentof the gene of claim
 2. 4. A nucleic acid that encodes a naturallyoccurring DapE protein of Helicobacter pylori and hybridizes with thenucleic acid of SEQ ID NO:1 under the stringency conditions of about 16hrs at about 65° C., about 5×SSC, about 0.1% SDS, about 2× Denhardt'ssolution, about 150 μg/ml salmon sperm DNA with washing at about 65° C.,30 min, 2×, in about 0.1×SSPE/0.1% SDS.
 5. A nucleic acid probe thathybridizes with the nucleic acid of SEQ ID NO:1 under the stringencyconditions of about 16 hrs at about 65° C., about 5×SSC, about 0.1% SDS,about 2× Denhardt's solution, about 150 g/ml salmon sperm DNA withwashing at about 65° C., 30 min, 2×, in about 0.1×SSPE/0.1% SDS.
 6. Anucleic acid primer that hybridizes with the nucleic acid of SEQ ID NO:1under the stringency conditions of 35 cycles of 94° C. for 1 min, 50° C.for 2 min, and 72° C. for 2 min, with a terminal extension at 72° C. for10 min.
 7. A purified mutant strain of Helicobacter pylori that does notexpress a functional DapE protein.
 8. The mutant strain of claim 7deposited with the American Type Culture Collection under accession no.ATCC
 55897. 9. The mutant Helicobacter pylori of claim 7, containing aplasmid comprising a nucleic acid encoding a functional DapE protein.10. The mutant strain of claim 7, having in its chromosome a nucleicacid encoding a foreign protein.
 11. The mutant strain of claim 10,wherein the foreign protein encoded is an immunogen of an infectiousbacterium, virus, yeast, fungus or parasite selected from the groupconsisting of Salmonella enteritidis, Shigella species, Yersinia,enterotoxigenic and enterohemorrhagic E. coli, Mycobacteriumtuberculosis, Streptococcus pyogenes, Bordatella pertussis, Bacillusanthracis, P. falciparum, human immunodeficiency virus, respiratorysyncytial virus, influenza virus, histoplasma capsulatum.
 12. The mutantstrain of claim 10, wherein the foreign protein encoded is a spermimmunogen.
 13. The mutant Helicobacter pylori of claim 10, containing aplasmid comprising a nucleic acid encoding a functional DapE protein.14. The mutant strain of claim 7, having a plasmid comprising a nucleicacid encoding a foreign protein.
 15. The mutant strain of claim 14,wherein the foreign protein encoded is an immunogen of an infectiousbacterium, virus, yeast, fungus or parasite selected from the groupconsisting of Salmonella enteritidis, Shigella species, Yersinia,enterotoxigenic and enterohemorrhagic E. coli, Mycobacteriumtuberculosis, Streptococcus pyogenes, Bordatella pertussis, Bacillusanthracis, P. falciparum, human immunodeficiency virus, respiratorysyncytial virus, influenza virus, histoplasma capsulatum.
 16. The mutantHelicobacter pylori of claim 14, containing a plasmid comprising anucleic acid encoding a functional DapE protein.
 17. A method ofmaintaining long term expression of a foreign antigen in Helicobacterpylori, comprising: a. transforming a mutant Helicobacter pylori ofclaim 7 with a plasmid comprising a nucleic acid encoding a functionalDapE protein comprising a nucleic acid encoding the foreign protein; andb. maintaining the mutant Helicobacter pylori from step a underconditions that permit expression of the foreign protein.
 18. A methodof maintaining the expression of a foreign protein in Helicobacterpylori, comprising: a. transforming a mutant Helicobacter pylori ofclaim 7 having in its chromosome a nucleic acid encoding the foreignprotein with a plasmid comprising a nucleic acid encoding a functionalDapE protein; b. maintaining the mutant Helicobacter pylori from step aunder conditions that permit expression of the foreign protein.
 19. Amethod of immunizing a subject against infection with Helicobacterpylori, comprising: a. administering to the subject the mutant strain ofclaim 7; b. supplementing the subject's diet with diaminopimelic acid tomaintain the mutant strain in the subject at least long enough for thesubject to mount an immune response to the strain; and c. ceasing thesupplementation of the subject's diet with diaminopimelic acid to killthe mutant strain in the immunized subject.
 20. A method of immunizing asubject against infection with a bacteria, virus, fungus or parasite,comprising: a) administering to the subject the mutant strain of claim11; b) supplementing the subject's diet with diaminopimelic acid tomaintain the mutant strain in the subject at least long enough for thesubject to mount an immune response to the foreign; and c) ceasing thesupplementation of the subject's diet with diaminopimelic acid to killthe mutant strain in the immunized subject.
 21. A method of immunizing asubject against infection with a bacteria, virus, fungus or parasite,comprising: a) administering to the subject the mutant strain of claim15; b) supplementing the subject's diet with diaminopimelic acid tomaintain the mutant strain in the subject at least long enough for thesubject to mount an immune response to the foreign protein; and c)ceasing the supplementation of the subject's diet with diaminopimelicacid to kill the mutant strain in the immunized subject.